Tyrosine Phosphorylation Of ZAP-70 Research Articles

Ssu72 phosphatase directly binds to ZAP-70, thereby providing fine-tuning of TCR signaling and preventing spontaneous inflammation

ZAP-70 is required for the initiation of T cell receptor (TCR) signaling, and Ssu72 is a phosphatase that regulates RNA polymerase II activity in the nucleus. However, the mechanism by which ZAP-70 regulates the fine-tuning of TCR signaling remains elusive. Here, we found that Ssu72 contributed to the fine-tuning of TCR signaling by acting as tyrosine phosphatase for ZAP-70. Affinity purification-mass spectrometry and an in vitro assay demonstrated specific interaction between Ssu72 and ZAP-70 in T cells. Upon TCR stimulation, Ssu72-deficient T cells increased the phosphorylation of ZAP-70 and downstream molecules and exhibited hyperresponsiveness, which was restored by reducing ZAP-70 phosphorylation. In vitro assay demonstrated that recombinant Ssu72 reduced tyrosine phosphorylation of ZAP-70 via phosphatase activity. Cd4-CreSsu72fl/fl mice showed a defect in the thymic development of invariant natural killer T cells and reductions in CD4+ and CD8+ T cell numbers in the periphery but more CD44hiCD62Llo memory T cells and fewer CD44loCD62Lhi naïve T cells, compared with wild-type mice. Furthermore, Cd4-CreSsu72fl/fl mice developed spontaneous inflammation at 6 mo. In conclusion, Ssu72 phosphatase regulates the fine-tuning of TCR signaling by binding to ZAP-70 and regulating its tyrosine phosphorylation, thereby preventing spontaneous inflammation.

Tumor Fibroblast-Derived FGF2 Regulates Expression of SPRY1 in Esophageal Tumor-Infiltrating T Cells and Plays a Role in T-cell Exhaustion.

T-cell exhaustion was initially identified in chronic infection in mice and was subsequently described in humans with cancer. Although the distinct signature of exhausted T (TEX) cells in cancer has been well investigated, the molecular mechanism of T-cell exhaustion in cancer is not fully understood. Using single-cell RNA sequencing, we report here that TEX cells in esophageal cancer are more heterogeneous than previously clarified. Sprouty RTK signaling antagonist 1 (SPRY1) was notably enriched in two subsets of exhausted CD8+ T cells. When overexpressed, SPRY1 impaired T-cell activation by interacting with CBL, a negative regulator of ZAP-70 tyrosine phosphorylation. Data from the Tumor Immune Estimation Resource revealed a strong correlation between FGF2 and SPRY1 expression in esophageal cancer. High expression of FGF2 was evident in fibroblasts from esophageal cancer tissue and correlated with poor overall survival. In vitro administration of FGF2 significantly upregulated expression of SPRY1 in CD8+ T cells and attenuated T-cell receptor-triggered CD8+ T-cell activation. A mouse tumor model confirmed that overexpression of FGF2 in fibroblasts significantly upregulated SPRY1 expression in TEX cells, impaired T-cell cytotoxic activity, and promoted tumor growth. Thus, these findings identify FGF2 as an important regulator of SPRY1 expression involved in establishing the dysfunctional state of CD8+ T cells in esophageal cancer. SIGNIFICANCE: These findings reveal FGF2 as an important regulator of SPRY1 expression involved in establishing the dysfunctional state of CD8+ T cells and suggest that inhibition of FGF2 has potential clinical value in ESCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5583/F1.large.jpg.

TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses.

Quantitative Phosphoproteome Analysis Unveils LAT as a Modulator of CD3ζ and ZAP-70 Tyrosine Phosphorylation

Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.

Visualizing the physiological self-reactivity of epidermal γδ TCR (P3280)

Abstract Dendritic epidermal T cells (DETCs) remain activated in vivo at steady state through their monomorphic Vgamma5-Vdelta1 TCR, forming phosphotyrosine-rich aggregates located on projections, called PALPs (Chodaczek et al. 2012). This recent discovery suggests the existence of beneficial self-reactivity on the part of the evolutionarily-preserved γδ TCR. Located at barrier-forming squamous keratinocyte junctions, and remarkably long-lived, the PALPs display multiple hallmarks of true immunological synapses, including TCR clustering and CD3-zeta and ZAP70 tyrosine phosphorylation. Using two independent super-resolution microscopy techniques: the structured illumination microscopy (SIM) and stimulated emission depletion (STED), we show that PALPs consist of several tens of peripherally distributed, tyrosine-phosphorylated TCR microclusters. Individual microclusters are uniformly sized at 150-160 nm and are associated with distinct signaling intermediates, suggesting that they represent supra-molecular signaling assemblies of a defined composition. Intravital fluorescence recovery after photobleaching and high speed time-lapse imaging showed that small intracellular cargo granules trafficked bi-directionally within T cell protrusions between large stationary granules and the PALPs. These studies reveal the PALPs as dynamically active TCR-activatory immunological synapses, and suggest that this novel form of physiological TCR self-reactivity mediates homeostatic secretory functions.

Abstract 911: Tyrosine kinase inhibitor Gefitinib selectivley induces apoptosis in Zap70+ chronic lymphocytic leukemia cells and inhibits B cell receptor signaling.

Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is characterized by an accumulation of B cells in the peripheral blood, lymph nodes, and bone marrow leading to immunosuppression. Patients can be stratified into two distinct groups based upon the expression of a T-cell receptor kinase, ZAP70. CLL patients with ≥20% cells staining for the ZAP70 have more aggressive disease and survive shorter times compared with patients with lower ZAP70 expression. Thus, tyrosine kinase inhibitors could be attractive therapeutic agents targeting ZAP70 over expressing CLL cells. In this study, we investigated tyrosine kinase inhibitor gefitinib that has been shown to inhibit tyrosine kinase Syk (a ZAP70 family member) and is not a myelo- or immuno-suppressive drug as other chemotherapeutic agents are in treating CLL. We found using MTT viability assay that the tyrosine kinase inhibitor gefitinib is effective in primary CLL cells with a significant preference for Zap70+ CLL; gefitinib had a median IC50 of 4.5 μM in Zap70+ patient cells and >15.0 μM in Zap70- patient cells. Flow cytometry analysis showed a decrease of viable Zap70+ CLL cells 24 hours post gefitinib treatment. Although gefitinib decreased the viability of the Zap70+ Jurkat T leukemia cell line, it failed to affect T cells from CLL patients. Western blot analysis showed gefitinib inhibited tyrosine phosphorylation of ZAP70 and Syk in CLL cells. In addition, both basal and B cell receptor-stimulated tyrosine phosphorylation was reduced after gefinitib treatment. Compared with other known inhibitors dasatinib and ibrutinib, gefitinib has the same downstream inhibition of Erk and Akt phosphorylation in Zap70+ cells. The significance of Zap70 expression was further determined by treating the Raji B lymphoma cell line transduced with vector control or vector encoding Zap70; indeed Zap70 expression sensitized the B cell line Raji to gefitinib treatment whereas other tyrosine kinase inhibitors were less effective. Therefore, gefitinib inhibits the Zap70 signaling pathway in CLL cells and holds potential as a targeted therapy for aggressive CLL. Citation Format: Rebecca Dielschneider, Wenyan Xiao, Ju Yoon Yoon, Edward Noh, James B. Johnston, Spencer B. Gibson. Tyrosine kinase inhibitor Gefitinib selectivley induces apoptosis in Zap70+ chronic lymphocytic leukemia cells and inhibits B cell receptor signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 911. doi:10.1158/1538-7445.AM2013-911

Abstract 5232: Tyrosine kinase inhibitor, gefitinib targets ZAP-70 over expressing chronic lymphocytic leukemia cells

Abstract Most patients with chronic lymphocytic leukemia (CLL) initially respond to chemotherapy but relapse. There is a subset of CLL patients that have aggressive disease characterized by an over expression of the tyrosine kinase ZAP-70. These ZAP-70+ patients have a poorer survival and are more resistant to chemotherapy. The biological effects of ZAP-70 in CLL are believed to be related to its ability to enhance activation of Syk with subsequent triggering of down-stream modulators, such as ERK and AKT. Normally, Syk is activated by increased phosphorylation by Lyn, which is increased in CLL, or by activation of the B-cell receptor. Gefitinib is a tyrosine kinase inhibitor that is presently used to treat lung cancer. The drug inhibits epidermal growth factor receptor (EGFR) tyrosine kinase activity but has activity against >20 other kinase targets, including members of the Src family of kinases, such as Lyn and Syk. We thus evaluated the cytotoxic activity of gefitinib against primary CLL cells using the MTT assay. Gefitinib was cytotoxic to ZAP-70+ (≥20% cells positive) CLL samples (median IC50, ∼3.0 µM) while ZAP-70- patients failed to respond to gefitinib with a median IC50 of >15.0 µM. Jurkat cells, which are T cells that express high levels of ZAP-70, were more sensitive to gefitinib than B cell lines that did not express ZAP-70 (BJAB and NALM6). Further studies confirmed that gefitinib was inducing cell death through apoptosis. Gefitinib was also effective against ZAP-70+ CLL cells that were resistant to fludarabine and chlorambucil indicating a novel mechanism of action to standard chemotherapy. Enhanced apoptosis was observed in ZAP70+ CLL and Jurkat cells exposed to gefitinib and fludarabine or chlorambucil, but this was not observed in ZAP-70- or the B cell lines. In contrast, another EGFR inhibitor, erlotinib, had no effect against ZAP-70+ CLL cells indicating that gefitinib was inhibiting a kinase unaffected by erlotinib. When Jurakat and ZAP-70+ CLL cells were treated with gefitinib, there was reduced total tyrosine phosphorylation and decreased tyrosine phosphorylation of ZAP-70, and Syk but no difference in Lyn phosphorylation. Furthermore, gefitinib blocked B cell receptor activation and mediated cell survival. Taken together, these results indicate that gefitinib may be useful agent in the treatment of ZAP-70+ CLL, either alone or in combination with fludarabine or chlorambucil. Ongoing studies are determining the precise mechanism of action of gefitinib in ZAP-70+ CLL and assessing its effects on the CLL microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5232. doi:1538-7445.AM2012-5232

Human CD38 Is a Potential Therapeutic Target for Sekected Chronic Lymphocytic Leukemia cases.

Chronic lymphocytic leukemia (CLL), the most common leukemia in Europe and North America, is characterized by a highly heterogeneous clinical behavior. Absence of mutations in the immunoglobulin variable (IgV) genes, expression of the surface receptor CD38 and the cytoplasmatic kinase ZAP-70 represent independent molecular markers of unfavorable prognosis.Recent data indicates that the proliferative core of the disease resides within peripheral lymphoid organs. There, the neoplastic cells have access to the antigen as well as to microenvironmental signals necessary to the maintenance and expansion of the clone. Our group has investigated the role of CD38 in CLL not only as a disease marker but also as a pathogenetic agent. We showed that CD38 ligation by agonistic monoclonal antibodies (mAbs) or the CD31 ligand is followed by proliferation and blast transformation of a subset of CLL cells. CD38/CD31 interactions occur predominantly in the lymph nodes and spleen where CD31 is abundantly expressed by stromal and nurse like cells (NLC). The signaling pathway initiated upon CD38 engagement relies upon tyrosine phosphorylation of ZAP-70 as a necessary step. CLL cells expressing CD38 and ZAP-70 also show the highest migration propensity towards CXCL12, a critical chemokine produced by NLC to attract CLL cells to lymph nodes. Collectively, these results point to a crucial role for the CD38/ZAP-70 pathway in directing CLL cells from the blood to peripheral lymphoid organs and suggest that targeting CD38 might impair CLL cells homing to sites favorable for neoplastic growth.In order to obtain a genome-wide signature of the transcriptional events taking place upon CD38/CD31 contacts CLL cells from 10 patients were co-cultured with CD31 transfected (L-CD31+) cells and L-mock controls for 5 days. Microarray analyses revealed 1,645 sequences selectively modulated as a consequence of CD38/CD31 interactions with 84 differentially regulated pathways: 12 of these pathways regulated cell adhesion/movement phenomena and 14 lymphocyte signalling.Attention was next focused on the impact of blocking anti-CD38 mAbs on CLL homing in vivo. To investigate the homing of CLL cells from blood to spleen and BM, PBMC were injected in the tail vein of female NOD/SCID mice and left to home for 24 hours. Next, mice were sacrificed and human cells tracked in spleen and BM by staining with specific reagents. Homing was significantly reduced when human cells were pre-incubated with anti-CD38 mAbs immediately prior to in vivo delivery. The in vivo data provided a rationale for a more in depth in vitro investigation of the effects of anti-CD38 mAbs on the CXCL12/CXCR4 signaling pathway. Results indicate that CXCL12-mediated chemotaxis can be blocked by anti-CD38 mAbs. Pre-treatment of CLL cells with anti-CD38 mAbs impairs CXCR4 signaling, as indicated by lack of Ca2+ mobilization, ERK1/2 activation and receptor internalization. These effects are specific for CD38+ samples, as no interference in migration was recorded when anti-CD38 mAbs were incubated with CD38− cells.The molecular explanation may be found at least in part in a physical proximity between CD38 and CXCR4 on the cell membrane, as shown by confocal microscopy and coimmunoprecipitation studies. In line with this working hypothesis, CXCL12 binding to CXCR4 is effectively inhibited following pre-incubation with anti-CD38 mAbs.In conclusion, these results offer biological evidence of a functional cross-talk between CD38 and CXCR4 signaling pathways. They also provide a preliminary rationale for the use of anti-CD38 ligands in therapy of a subset of CLL patients, representing a novel approach interfering with deleterious growth circuits and increasing the susceptibility of leukemic cells to conventional chemotherapy.

CD38 AND ZAP‐70 REGULATE CXCL12‐MEDIATED CHEMOTAXIS OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS

Absence of mutations in the IgV genes, expression of the receptor CD38 and of the kinase ZAP‐70 are independent markers of unfavorable outcome in CLL patients.Our group studies the role of CD38 in CLL not only as disease marker but also as pathogenetic agent. We showed that CD38 enhances proliferation of CLL cells through binding CD31, the ligand expressed by stromal and endothelial cells. Further, i) CD38+/ZAP‐70+ cells efficiently migrate towards CXCL12, ii) CD38 engagement leads to tyrosine phosphorylation of ZAP‐70 and iii) CD38 pathway is impaired in CD38+/ZAP‐70− CLL cells.Collectively, these data point to a role for the CD38/ZAP‐70 pathway in CLL migration towards CXCL12. Indeed, CXCL12‐mediated chemotaxis can be blocked by anti‐CD38 mAbs. Pre‐treatment of CLL with anti‐CD38 mAbs impairs CXCR4 signaling, as indicated by lack of Ca2+ mobilization, ERK1/2 activation and receptor internalization. Further, CD38 and CXCR4 are associated, as shown by confocal microscopy and co‐immunoprecipitation.In conclusion, these results offer biological evidence explaining why the combination of CD38 and ZAP‐70 results in a more efficient identification of CLL patients with aggressive disease. Further, they provide preliminary rationale for using anti‐CD38 ligands or inhibitors as adjunct therapeutic tools.

CXCR3‐mediated T‐cell chemotaxis involves ZAP‐70 and is regulated by signalling through the T‐cell receptor

The chemokine receptor CXCR3 is critical for the function of activated T cells. We studied the molecular mechanisms of CXCR3 signalling. The addition of CXCR3 ligands to normal human T cells expressing CXCR3 led to the tyrosine phosphorylation of multiple proteins. Addition of the same ligands to Jurkat T cells engineered to express CXCR3 induced tyrosine phosphorylation of proteins with molecular weights similar to those in normal cells. Immunoblotting with phosphotyrosine-specific antibodies identified Zeta-associated protein of 70,000 molecular weight (ZAP-70), linker for the activation of T cells (LAT), and phospholipase-C-gamma1 (PLCgamma1) to be among the proteins that become phosphorylated upon CXCR3 activation. ZAP-70 was phosphorylated on tyrosine 319, LAT on tyrosines 171 and 191, and PLCgamma1 on tyrosine 783. The ZAP-70 inhibitor piceatannol reduced CXCR3-mediated tyrosine phosphorylation of ZAP-70, LAT, PLCgamma1 and mitogen-activated protein kinase Erk and it reduced CXCL10-mediated chemotaxis of both CXCR3-transfected Jurkat T cells and normal T cells expressing CXCR3. These results are consistent with the involvement of ZAP-70 in CXCR3-mediated protein tyrosine phosphorylation and CXCR3-induced T-cell chemotaxis. Studies with the Lck-deficient Jurkat T-cell line, JCAM1.6, demonstrated that phosphorylation of ZAP-70 after CXCR3 activation is a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function.

Intermolecular relations between the glucocorticoid receptor, ZAP-70 kinase, and Hsp-90

The glucocorticoid receptor (GR) participates in both genomic and non-genomic glucocorticoid hormone (GC) actions by interacting with other cytoplasmic signalling proteins. Previously, we have shown that high dose Dexamethasone (DX) treatment of Jurkat cells causes tyrosine phosphorylation of ZAP-70 within 5 min in a GR-dependent manner. By using co-immunoprecipitation and confocal microscopy, here we demonstrate that the liganded GR physically associates with ZAP-70, in addition to its phosphorylation changes. The association of the ligand-bound GR and ZAP-70 was also observed in HeLa cells transfected with ZAP-70, suggesting that this co-clustering is independent of lymphocyte specific factors. Furthermore, the ZAP-70 was found to also co-precipitate with Hsp-90 chaperone both in Jurkat and transgenic HeLa cells, independent of the presence of DX. These findings raise the possibility that ZAP-70 may serve as an important link between GC and TcR-induced signaling, thereby transmitting non-genomic GC action in T-cells.

TULA: A novel protein that affects activation and degradation of protein tyrosine kinases

TULA (T-cell Ubiquitin Ligand) is a novel protein containing UBA and SH3 domains; and the C-terminal half contains homology to PhosphoGlycerate Mutases. TULA is the first protein described to contain both the UBA and SH3 domains. TULA is a primarily lymphoid protein, although the second protein of its family is ubiquitously present. TULA was shown to bind c-Cbl, ubiquitin and ubiquitylated proteins. As c-Cbl is an E3 ubiquitin ligase and is known to downregulate activated protein tyrosine kinases (PTKs) by ubiquitylation, involvement of TULA in this process was studied. It was found that TULA protects receptor PTKs from c-Cbl-dependant ubiquitylation driven degradation. Mice lacking TULA and the second protein of this family were viable and normal. The double knockout T cells had enhanced proliferative ability. Further analysis of these T cells showed increased tyrosine phosphorylation of Zap-70, a Syk family kinase. Therefore, TULA proteins exert an effect on PTKs but its molecular mechanism is not known. Hence, to understand these molecular mechanisms we chose to study the effect of TULA on Syk, a non-receptor PTK. Syk is found in both B and T cells, binds to c-Cbl and is ubiquitylated by c-Cbl. Our studies have shown that TULA activates Syk. However, the effect of the second protein is opposite from TULA’s effect on Syk. Therefore, in contrast to knockout data, which suggest that both the members of this family have similar/overlapping effects, our studies show that these proteins may have different effects and act through different functional mechanisms. [NIH Grant, Dept. funds]

Imatinib mesylate suppresses cytokine synthesis by activated CD4 T cells of patients with chronic myelogenous leukemia

Although imatinib mesylate (IM) is highly effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia (CML), it is known to suppress T-cell proliferation in vitro. As cytokines are required for T-cell proliferation, we investigated the effects of IM on cytokine synthesis by T cells of CML patients by assessing cytokine synthesis by activated CD4+ and CD8+ T cells in vitro. The activation of T cells in the whole blood of IM-treated patients (CML-IM) with Staphylococcus enterotoxin B resulted in significantly lower percentages of CD4+ T cells that synthesized interleukin 2 (P = 0.017), interferon-gamma (P = 0.010), and tumor necrosis factor-alpha (P = 0.009) than did the activated T cells of control subjects. The addition of exogenous IM to the cultures of peripheral blood mononuclear cells of CML-IM patients reduced Th1 cytokine synthesis by the CD4+ T cells. Furthermore, IM therapy at clinical doses suppressed the tyrosine phosphorylation of ZAP70. These findings suggest that inhibition of ZAP70 signaling pathway and suppression of Th1 cytokine synthesis by CD4+ T cells required the presence of IM at the time of T-cell activation through the T-cell receptor.

Imatinib inhibits T-cell receptor–mediated T-cell proliferation and activation in a dose-dependent manner

AbstractThe tyrosine kinase inhibitor imatinib (imatinib, STI571, Glivec, and Gleevec) is increasingly used in patients undergoing allogeneic transplantation for leukemia. However, little is known regarding its potential immunoregulatory effects. Here, we investigate the effect of imatinib on T-cell receptor (TCR)–mediated activation of human T cells. Following stimulation with the anti-CD3 antibody 12F6, proliferation of activated T cells was almost completely inhibited by 10 μM imatinib. Furthermore, antigen-triggered expansion of CD8+ T cells in response to immunodominant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) peptides was significantly reduced. Up-regulation of the activation markers CD25 and CD69 in response to TCR cross-linking was suppressed by imatinib at a mean inhibitory concentration 50% (IC50) of 5.4 μM and 7.3 μM, respectively; interleukin 2 (IL-2) production was also impaired. Analysis of the TCR-induced signaling cascade showed that imatinib substantially reduced tyrosine phosphorylation of ZAP70 and LAT in response to activation through the TCR. Sequence comparisons of all 90 tyrosine kinase genes in the human genome for homology in the adenosine triphosphate (ATP) binding pocket identified LCK, which is required for ZAP70 activation, as a likely target for imatinib. The IC50 for LCK inhibition by imatinib was 0.6 μM to 0.8 μM in an in vitro tyrosine kinase assay. In summary, imatinib can interfere with T-cell activation in vitro, and its impact on the frequency of opportunistic infections and graft-versus-host or graft-versus-leukemia reactions after transplantation should be investigated in clinical trials.

CD38 Ligation in B-Chronic Lymphocytic Leukemia Cells Induces Sequential Tyrosine Phosphorylation of ZAP70, PLC- γ2 and ERK1/2 Proteins.

CD38 is a negative prognostic marker in B-chronic lymphocytic leukemia (B-CLL). In a previous study we observed that CD38 and IL-2 cooperate in inducing proliferation and survival of B-CLL cells. Now attention is given to the signaling pathway ruled by CD38 in B-CLL cells. We show that CD38 ligation triggers a prominent tyrosine phosphorylation of the ZAP70 kinase in 6 out of 9 patients studied (2 representative shown in figure 1). ZAP70 activation is evident 1 minute after receptor cross-linking and tends to diminish within 5 minutes. Tyrosine phosphorylation of syk is undetectable in these conditions. A functional relationship between CD38 and ZAP70 was confirmed by the analysis of a panel of B cell lines. Indeed, CD38 ligation in Nalm-6, a pre-B cell line identified as ZAP70+, wais followed by ZAP70 tyrosine phosphorylation, with a kinetics overlapping the one observed with the B-CLL samples.Downstream events include the sequential activation of BTK and PLC-γ2, the latter likely responsible for the cytoplasmic Ca2+ mobilization, observed both in B-CLL and in the Nalm-6 cells. The signaling events culminate with the activation of ERK1/2, but not of SAP/JNK and p38 proteins. The results obtained with purified B-CLL cells from 2 representative patients are shown in figure 1.Together, these data i) highlight for the first time a functional relationship between CD38 and ZAP70 in B cells and ii) identify crucial cytoplasmic steps in the signaling cascade promoted by CD38, thus providing a mechanistic base to explain why B-CLL patients with a CD38+/ZAP70+ clone are characterized by the most unfavourable clinical course. Selected bibliography [Display omitted]

Proliferation of CD8+ T-Cells Specific for CMV and EBV in Response to TCR Mediated Recognition of Cognate Antigen Is Inhibited by Imatinib in a Dose-Dependent Manner.

We and others have shown that the tyrosine kinase inhibitor imatinib (STI571, Gleevec®) inhibits T-cell proliferation and activation at concentrations achieved in vivo. At 10μM, imatinib inhibited T-cell receptor (TCR)-mediated proliferation of purified peripheral blood T-cells almost completely. Up-regulation of the activation markers CD25 and CD69 at 24h in response to TCR cross-linking was suppressed by imatinib at a mean IC50 of 5.4μM and 7.3μM, respectively and IL-2 production was also severely impaired. However, these assays may not fully reflect the response to clinical relevant antigens. Therefore, we chose to investigate the antigen-triggered proliferation of memory CD8+ T-cells specific for immunodominant CMV and EBV HLA-A2 peptide epitopes. We used HLA-peptide tetramers to identify healthy blood donors with detectable CMV- or EBV-specific CD8+ T-cell populations. Purified T-cells from these donors were then stimulated with the CMV peptide pp65495–503 or the EBV peptide BMFLI259–267. Antigen-induced proliferation was measured by dilution of the vital dye CFSE over a period of 4 or 8 days. The magnitude of the virusspecific CD8+ T-cell population ranged from 0.5 % to 7.1% of CD8+ T-cells for CMV and from 0.05% to 0.35% of CD8+ T-cells for EBV. Antigen-specific CD8+ T-cells from all 10 donors studied proliferated in response to the CMV peptide. In 8 from 10 donors, imatinib reduced CMV peptide induced proliferation. With increasing imatinib concentrations (range: 5 – 10μM), we observed dose dependent reduction of both the number of cells undergoing cell division and the average number of divisions completed per cell. Comparable inhibition of specific T-cell proliferation in response to the EBV-derived peptide was observed in two donors. Immunoblots demonstrated that imatinib substantially reduced tyrosine phosphorylation of ZAP70 and LAT in response to TCR-mediated activation in Jurkat T-cells. Sequence comparisons of all 90 tyrosine kinase genes in the human genome for homology in the ATP binding pocket identified Lck, which is required for ZAP70 activation, as a likely target for imatinib. Our results indicate that imatinib may interfere with clinically important T-cell effector functions. As concentrations sufficient for half-maximal inhibition of TCR signalling are achieved in vivo, imatinib could increase the risk of opportunistic infections and impact on GVH and GVL reactions post-transplantation especially when used in conjuction with other immunosuppressive agents. Therefore, close monitoring of patients on imatinib for CMV reactivation or EBV-induced lymphoproliferative diseases, especially in stem cell transplant recipients, appears warranted.

Interaction of SAP-1, a Transmembrane-type Protein-tyrosine Phosphatase, with the Tyrosine Kinase Lck: ROLES IN REGULATION OF T CELL FUNCTION

SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function.

Altered proximal T-cell receptor signalling events in mouse CD4+ T cells in the presence of anti-CD4 monoclonal antibodies: evidence for reduced phosphorylation of Zap-70 and LAT.

Anti-CD4 monoclonal antibodies are potential therapeutic agents for the prevention of autoimmune disease and treatment of rejection after organ transplantation and are capable of both restoring tolerance to self-antigens and inducing tolerance to antigens introduced under the cover of the antibody therapy in vivo. Tolerance to donor alloantigens can be induced in vivo by administering donor alloantigen in combination with either depleting (YTA 3.1) or nondepleting (YTS 177) anti-CD4, 28 days before heart transplantation in the mouse. The effect of anti-CD4 on proximal T-cell receptor (TCR) signalling pathways and proliferation was investigated in vitro and in vivo in the presence and absence of YTA 3.1 or YTS 177. Anti-CD4 was found to perturb proximal signalling events upon TCR/CD3 ligation, resulting in reduced tyrosine phosphorylation of Zap-70 and LAT (linker for activation of T cells) and reduced association of tyrosine-phosphorylated LAT with lck. This ultimately resulted in severely reduced proliferation of the responding CD4+ T cells. The signalling profile of the anti-CD4-treated cells resembled that of anergic T cells. This could be a result of a common mechanism involving perturbation in the formation of the central supramolecular activation cluster of the immunological synapse by impaired recruitment of CD4 and CD28, thereby resulting in severely reduced lck activation.

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways.

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.

Human CD38 and CD16 are functionally dependent and physically associated in natural killer cells

CD38, a surface glycoprotein of unrestricted lineage, is an ectoenzyme (adenosine diphosphate [ADP] ribosyl cyclase/cyclic ADP-ribose hydrolase) that regulates cytoplasmic calcium. The molecule also performs as a receptor, modulating cell-cell interactions and delivering transmembrane signals, despite showing a structural ineptitude to the scope. CD38 ligation by agonistic monoclonal antibodies induced signals leading to activation of the lytic machinery of natural killer (NK) cells from adults; similar signals could not be reproduced in YT and NKL, 2 CD16(-) human NK-like lines. It was hypothesized that CD38 establishes a functional cooperation with professional signaling molecules of the NK cell surface. The present work answers the question about the molecule exploited by CD38 for signaling in NK cells, using as a model CD16(-) NK lines genetically corrected for CD16 expression. Our results indicate that a functional CD16 molecule is a necessary and sufficient requisite for CD38 to control an activation pathway, which includes calcium fluxes, tyrosine phosphorylation of ZAP70 and mitogen-activated protein kinase, secretion of interferon-gamma, and cytotoxic responses. Fluorescence resonance energy transfer and cocapping experiments also showed a surface proximity between CD38 and CD16. These results were confirmed by using the NKL cell line, in which CD16(+) and CD16(-) variants were obtained without genetic manipulation. Together, our findings show CD38 to be a unique receptor molecule that cannot signal by itself but whose receptor function is rescued by functional and physical associations with a professional signaling structure that varies according to lineage and environment. This molecule is CD16 in NK cells.