ZAP70
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T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- aminokyselinové motivy MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- Jurkat buňky MeSH
- lidé MeSH
- membránové proteiny chemie metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- prolin analýza MeSH
- protein-tyrosinkináza ZAP-70 metabolismus MeSH
- receptory antigenů T-buněk metabolismus MeSH
- thymus imunologie MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Patients with unmutated IGHV (U-CLL) usually progress rapidly, whereas patients with mutated IGHV (M-CLL) have a more indolent disease. The expression of several genes correlates closely with the IGHV mutational status and could be used to assess prognosis in CLL. We analyzed the prognostic relevance of COBLL1, LPL, and ZAP70 gene expression, which correlated with IGHV mutational status (p < 0.0001), in 117 CLL patients and established a prognostic parameter dividing the tested cohort according to the disease aggressiveness. Our prognostic parameter was validated on an independent cohort of 161 CLL patients and achieved a high accuracy (94%). Patients divided according to the prognostic parameter differ in overall survival and time to first treatment (p < 0.0001, HR = 2.300/5.970, 95% CI: 1.587-3.450/4.621-15.86). Our approach provides a reliable alternative method to prognosis assessment via IGHV mutational status analysis.
- MeSH
- analýza přežití MeSH
- chronická lymfatická leukemie diagnóza genetika mortalita MeSH
- dospělí MeSH
- exprese genu MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipoproteinlipasa genetika MeSH
- mutace * MeSH
- nádorové biomarkery MeSH
- prognóza MeSH
- progrese nemoci MeSH
- protein-tyrosinkináza ZAP-70 genetika MeSH
- reprodukovatelnost výsledků MeSH
- ROC křivka MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- transkripční faktory genetika MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.
- MeSH
- aminokyselinové motivy MeSH
- buněčná membrána metabolismus MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- cytosol metabolismus MeSH
- difuze MeSH
- fluorescenční spektrometrie MeSH
- fosforylace MeSH
- Jurkat buňky MeSH
- lidé MeSH
- optogenetika MeSH
- receptory antigenů T-buněk chemie metabolismus MeSH
- shluková analýza MeSH
- signální transdukce * MeSH
- světlo MeSH
- tyrosinkinasa p56(lck), specifická pro lymfocyty metabolismus MeSH
- vápník metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal expansion of mature B-lymphocytes. Mutational status of the immunoglobulin variable heavy chain region (IGHV) gene stratifies CLL patients into two prognostic groups. We performed microarray analysis of CLL cells using the Agilent platform to detect the most important gene expression differences regarding IGHV status in CLL cells. We analyzed a cohort of 118 CLL patients with different IGHV mutational status and completely characterized all described prognostic markers using expression microarrays and quantitative real-time RT-PCR (reverse transcription PCR). We detected lymphocyte-activation gene 3 (LAG3) as a novel prognostic marker: LAG3 high expression in CLL cells correlates with unmutated IGHV (P < 0.0001) and reduced treatment-free survival (P = 0.0087). Furthermore, quantitative real-time RT-PCR analysis identified a gene-set (LAG3, LPL, ZAP70) whose overexpression is assigned to unmutated IGHV with 90% specificity (P < 0.0001). Moreover, high expression of tested gene-set and unmutated IGHV equally correlated with reduced treatment-free survival (P = 7.7 * 10(-11) vs. P = 1.8 * 10(-11)). Our results suggest that IGHV status can be precisely assessed using the expression analysis of LAG3, LPL, and ZAP70 genes. Expression data of tested markers provides a similar statistical concordance with treatment-free survival as that of the IGHV status itself. Our findings contribute to the elucidation of CLL pathogenesis and provide novel prognostic markers for possible application in routine diagnostics.
- MeSH
- CD antigeny genetika MeSH
- chronická lymfatická leukemie genetika mortalita MeSH
- dospělí MeSH
- geny pro těžké řetězce imunoglobulinů genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipoproteinlipasa genetika MeSH
- míra přežití MeSH
- mutace MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein-tyrosinkináza ZAP-70 genetika MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chronická B-lymfocytární leukemie (B-CLL) představuje v našich zeměpisných oblastech nejčastěji diagnostikovanou leukemickou lymfoproliferaci dospělého věku. Přesné a jednoznačné určení diagnózy B-CLL s pomocí moderních technik imunocytologie má zásadní význam pro její odlišení od dalších subtypů leukemizovaných B-buněčných lymfoproliferací. Stanovení široké škály prognostických parametrů u pacientů s nízce a středně pokročilou chorobou poskytuje nezbytný rámec k úvaze o vhodnosti a intenzitě vedené léčby při použití řady nových terapeutických možností. Stanovení procenta mutací IgVH genů, ZAP 70, exprese CD38, chromozomálních změn a mutací genu p53 představují v dnešní době nejmodernější nástroje k poznání biologického chování a potencionální agresivity leukemického klonu.
B-cell chronic lymphocytic leukemia (B-CLL) represents most common leukemia in Western Europe and North America adult population. Accurate diagnosis of the disease based on leukemic cell immunophenotype analysis has principal importance in differential diagnostic distinction of B-CLL from other leukemized B-cell malignancies. Determination of strong and independent prognostic parameters in early and intermediate disease stage patients could lead to consideration of appropriate therapeutic approach. Assessment of IgVH mutational status, ZAP70 and CD38 expression levels, genetic aberrations and p53 gene mutations constitute modern tools for understanding of biologic behavior and potential aggressiveness of leukemic clone.
The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).
- MeSH
- HLA-DR antigeny analýza MeSH
- imunofenotypizace metody MeSH
- kojenec MeSH
- lidé MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- primární imunodeficience diagnóza imunologie MeSH
- průtoková cytometrie metody MeSH
- T-lymfocyty imunologie MeSH
- těžká kombinovaná imunodeficience imunologie MeSH
- thymus imunologie MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
