mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse.

The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation-dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify mDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for mDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.

Towards a surrogate system to express human lipid binding TCRs.

BACKGROUND: Previously we reported that natural nut lipids were necessary for sensitization and that natural killer T cells (NKTs) must play a critical role in the development of food allergic responses. A major bottleneck in further understanding the interaction of nut lipids with the cells of the human immune system is the lack of well-characterized lipid responsive human cell lines. OBJECTIVE: In the present study, we engineered human T cell receptor (TCR) sequences TRAV10 and TRBV25 responsive to α-GalCer into a stable murine iNKT hybridoma and surrogate human T cell lines. RESULTS: The murine hybridoma system has shown to be problematic. To overcome this limitation, the expression of human TCR α/ß sequences has been achieved driven by a bidirectional promoter on a plasmids or a lentivirus system, employing stable DC cell lines as lipid presenting cells, and a stable T cell line as a surrogate system. Further, a commercial human Jurkat T cell line containing an inducible secreted luciferase reporter construct was shown to be functional and can be used for a transient expression of human TCRs in a lipid screening program. The transfection efficiencies were improved using the lentivirus polycistronic constructs containing the P2A sequence in a TCR αß/γδ null cell line (Jurkat 76). CONCLUSIONS: The results suggest that the mis-pairing of the endogenous α/ß TCR during ER folding in the presence of the new human TCR sequences could be impairing the functionality of the TCR lipid receptors. The surrogate systems presented here are important first steps in the establishment of human cell-specific lipid responsive libraries for the study of natural lipid substances.

ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

Ionizing Radiation Induces Morphological Changes and Immunological Modulation of Jurkat Cells.

Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca2+, an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca2+ sensitive K+ channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca2+ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca2+-dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients.

[Advances in application of Jurkat cell model in research on infectious diseases].

Infectious diseases can be caused by multiple pathogens, which can produce specific immune response in human body. The immune response produced by T cells is cellular immunity, which plays an important role in the anti-infection process of human body, and can participate in immunological protection and cause immunopathology. The outcome of various infectious diseases is closely related to cellular immune function, especially the function of T cells. Jurkat cells belong to the human acute T lymphocyte leukemia cell line. Jurkat cell model can simulate the function T lymphocytes, so it is widely used in the in vitro studies of T cell signal transduction, cytokines, and receptor expression, and can provide reference and guidance for the treatment of various infectious diseases and the research on their pathogenesis. The Jurkat cell model has been widely used in the in vitro studies of viral diseases and atypical pathogens, but parasitic infection studies using the Jurkat cell model are still rare. This article reviews advances in the application of Jurkat cell model in the research on infectious diseases.

Drebrin Regulation of Calcium Signaling in Immune Cells.

Store-operated Ca2+ channels are plasma membrane channels that are activated by depletion of intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+; however, little is known about their regulation. Our work has shown that the immunosuppressant compound BTP2, which blocks Ca2+ influx into cells, interacts with the actin-reorganizing protein, drebrin. Here we review the role of drebrin in the regulation of calcium signaling, with a focus on immune cells.

A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

In Vitro Effects of Thymoglobulin in Human Embryonic Kidney Cell Line (HEK293) in Culture.

BACKGROUND: Anti-thymocyte globulins are polyclonal T-cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobulin is purified rabbit immunoglobulin (Ig)G, obtained by immunization of rabbits with fetal human thymus, which depletes T lymphocytes by complement-dependent lysis and apoptosis, reduces production of cytokines, and decreases expression of adhesion molecules in endothelial cells. METHODS: To determine possible direct effects of Thymoglobulin on kidney cells during transplantation, we used the Human Embryonic Kidney cell line (HEK293) in culture. We measured membrane potential of the cells by use of the slow whole patch-clamp technique. We determined effects of Thymoglobulin on cell death and proliferation during hypoxia/re-oxygenation injury, using a hypoxic chamber. RESULTS: Depolarizations of HEK293 cells caused by Thymoglobulin were concentration-dependent and membrane potential-dependent, showing direct effects of Thymoglobulin on the HEK293 cells, whereas rabbit anti-thymocyte globulin produced against Jurkat cells (ATG-F) and normal rabbit IgG had no effects. To determine the effects of Thymoglobulin in hypoxia/re-oxygenation conditions, cells were incubated for 24 hours with Thymoglobulin in an atmosphere with 5% CO2-95% N2 at 37°C followed by 1 hour in atmosphere with 5% CO2-95% air at 37°C. The effects of hypoxia/re-oxygenation were detected by calculating cell death and determining the cell growth, using scratch test. CONCLUSIONS: Thymoglobulin prevented the cell death induced by hypoxia and re-oxygenation conditions. In addition, it accelerated the cell growth (improved scratch wound-healing). This is the first study to show the direct effects of Thymoglobulin on kidney-derived epithelial cells, which may lead to better understanding of its effects in kidney transplantation.

T cell adhesion triggers an early signaling pole distal to the immune synapse.

The immunological synapse forms at the interface between a T cell and an antigen-presenting cell after foreign antigen recognition. The immunological synapse is considered to be the site where the signaling cascade leading to T lymphocyte activation is triggered. Here, we show that another signaling region can be detected before formation of the synapse at the opposite pole of the T cell. This structure appears during the first minute after the contact forms, is transient and contains all the classic components that have been previously described at the immunological synapse. Its formation is independent of antigen recognition but is driven by adhesion itself. It constitutes a reservoir of signaling molecules that are potentially ready to be sent to the immunological synapse through a microtubule-dependent pathway. The antisynapse can thus be considered as a pre-synapse that is triggered independently of antigen recognition.

Anti-tumor effects of anti-T-cell globulin.

In vivo T-cell depletion using anti-T-cell antibodies is a standard procedure during allogeneic hematopoietic stem cell transplantation (allo-HSCT). Clinical data demonstrate that in vivo T-cell depletion with the anti-CD52 monoclonal antibody Alemtuzumab is associated with increased relapse rates of hematologic malignancies after allo-HSCT, underlining the importance of donor T cells for graft versus tumor activity. In contrast, recent results suggest that in vivo T-cell depletion with rabbit anti-T-cell globulin (ATG) Fresenius is not associated with tumor relapse after allo-HSCT, raising the possibility that ATG mediates antitumor effects. However, data on ATG's ability to bind to tumor cells and on its effect on the induction of antibody-dependent cellular cytotoxicity (ADCC) are lacking. We used ATG Fresenius, which contains polyclonal rabbit IgG directed against the human T-lymphoma cell line Jurkat, to study relevant mechanisms of ATG-mediated antitumor effects, including ADCC, complement-dependent cytotoxicity, and induction of apoptosis. Based on the knowledge that Jurkat cells aberrantly express myeloid markers and B-cell markers, we hypothesized that rabbit ATG Fresenius binds to a variety of hematologic malignancies. We found that ATG specifically binds to a variety of hematologic malignancies including acute myeloid leukemia and B-cell lymphoma in a concentration-dependent manner. We demonstrate that ATG mediates antitumor activity, including induction of ADCC, complement-dependent cytotoxicity, and apoptosis, toward different hematologic malignancies. Our results contribute to a better understanding of the effects of ATG on posttransplant immunology in patients undergoing allo-HSCT.

Comparison of rabbit antithymocyte globulin and Jurkat cell-reactive anti-T lymphocyte globulin as a first-line treatment for children with aplastic anemia.

The purpose of this study was to investigate the effects of rabbit antihuman thymocyte globulin (R-ATG) and Jurkat cell-reactive anti-T lymphocyte globulin (ATG-F) in the treatment of childhood aplastic anemia (AA) and compare their efficacy and side effects. A total of 53 children with AA were analyzed in the present study, including 32 cases of severe AA, 10 cases of very severe AA and 11 cases of transfusion-dependent nonsevere AA. While receiving immunosuppressive therapy (IST), 29 and 24 patients, all of whom received long-term oral supplement with cyclosporin A (CSA), androgen, and traditional Chinese medicines, were treated with R-ATG and ATG-F, respectively. If necessary, the patients were also given supportive care such as component transfusion and/or infection control. Absolute counts of peripheral blood lymphocyte at various time points were dynamically measured after ATG therapy. According to the International AA Treatment and Effect standards, we found that there were no statistically significant differences in the response rate (70.83% vs. 68.97%, p > 0.05) and the overall survive rate (83.33% vs. 82.76%, p > 0.05) between the ATG-F and R-ATG groups. In addition, no obvious differences were observed between these two groups in the response time, efficacy in severe AA and very severe AA, or the incidence rates of ATG-related adverse reactions. After ATG treatment, the extent of peripheral blood lymphocyte reduction and duration in peripheral blood were similar between the ATG-F and R-ATG groups. The results of this study showed that ATG-F and R-ATG had similar efficacy and adverse reactions in the first-line treatment of childhood AA, despite being derived from different immunogens.

High glucose driven expression of pro-inflammatory cytokine and chemokine genes in lymphocytes: molecular mechanisms of IL-17 family gene expression.

High glucose is an independent risk factor that alters the expression pattern of cytokines/chemokine leading to leukocyte activation in diabetes. Fluctuation of cytokine milieu in lymphocytes may lead to differentiation into a particular subset. Our objectives were to profile high glucose induced inflammatory gene expression in lymphocytes, to examine in vivo relevance in diabetes and to identify the key transcription factors and signaling pathways involved. Cytokine gene arrays and T-helper (Th1/Th2/Th17) cytokine profiler RT(2)-PCR arrays used for cytokine expression profiling followed by validation using Real Time-qPCR and relative RT-PCR in Jurkat T-lymphocytes, peripheral blood lymphocytes (PBLCs) from normal and diabetes subjects. Luciferase reporter plasmid, pharmacological inhibitors and mutant plasmids were used for promoter activation and signaling pathway studies. High glucose induced gene profiling in Jurkat T-lymphocytes showed significantly increased expression of 64 proinflammatory genes including IL-6 and IL-17A and most of these genes were Nuclear Factor (NF)-κB and AP-1 regulated. RT(2)-PCR array results suggested the transcriptional activation of IL-17 and its downstream signaling in Jurkat T-lymphocytes upon high glucose treatment. Candidate genes like Interleukin (IL)-17A, IL-17E IL-17F and IL-6 were up-regulated in both Jurkat T-lymphocytes and PBLCs from normal and diabetes subjects. This high glucose induced cytokine expression was due to promoter activation. Pharmacology inhibitor studies showed the involvement of NF-κB, protein kinase-C, p38 Mitogen activated protein kinase; Janus activated kinase-signal transducer and activator of transcription and extracellular regulated kinase signaling pathways. Further, high glucose treatment increased the adhesion of lymphocytes to human umbilical vein endothelial cells. These results show that IL-17 cytokines are induced by high glucose via key signaling pathways leading to lymphocyte activation and relevant to the pathogenesis of diabetic complications like atherosclerosis.

Aplotaxene blocks T cell activation by modulation of protein kinase C-θ-dependent pathway.

Aplotaxene, (8Z, 11Z, 14Z)-heptadeca-1, 8, 11, 14-tetraene, is one of the major components of essential oil obtained from Inula helenium root, which is used in Oriental medicine. However, the effects of aplotaxene on immunity have not been investigated. Here, we show that aplotaxene inhibits T cell activation in terms of IL-2 and CD69 expression. Aplotaxene, at a concentration that optimally inhibits IL-2 production, has little effect on apoptotic or necrotic cell death, suggesting that apoptosis is not a mechanism for aplotaxene-mediated inhibition of T cell activation. Aplotaxene affects neither superantigeninduced conjugate formation between Jurkat T cells and Raji B cells nor clustering of CD3 and LFA-1 at the immunological synapse. Aplotaxene significantly inhibits PKC-θ phosphorylation and translocation to the immunological synapse, and blocks PMA-induced T-cell receptor internalization. Furthermore, aplotaxene leads to inhibition of mitogen-activated protein kinases (JNK, ERK and p38) phosphorylation and NF-κB, NF-AT, and AP-1 promoter activities in Jurkat T cells. Taken together, our findings provide evidence for the immunosuppressive effect of aplotaxene on activated T cells through the modulation of the PKC-θ and MAPK pathways, suggesting that aplotaxene may be a novel immunotherapeutic agent for immunological diseases related to the overactivation of T cells.

Identification of functionally relevant phoshorylatable serine clusters in the cytoplasmic region of the human CD6 lymphocyte surface receptor.

CD6 is a transmembrane receptor expressed by all T and a subset of B lymphocytes, where it physically associates with the antigen-specific receptor to modulate activation and differentiation processes through still poorly understood mechanisms. Its cytoplasmic tail lacks intrinsic catalytic activity but presents several consensus motifs for phosphorylation. The present work reports on the identification of two constitutively phosphorylated serine clusters (S480/482/484 and S560/562/565/567/568), which are embedded into Casein Kinase 2 consensus motifs, and are indispensable for proper mitogen-activated protein kinase activation following CD6 ligation. The data point to a novel level of regulation of CD6 function by intracytoplasmic serine phosphorylation.

Anti-inflammatory properties of potato glycoalkaloids in stimulated Jurkat and Raw 264.7 mouse macrophages.

AIMS: The potato glycoalkaloids, α-chaconine, α-solanine and solanidine, along with potato peel extracts were investigated for potential anti-inflammatory effects in vitro. Their potential to reduce two biomarkers of inflammation, cytokine and nitric oxide (NO) productions, were assessed in the stimulated Jurkat and macrophage models, respectively. MAIN METHODS: Cytokine and nitric oxide productions were stimulated in Jurkat and Raw 264.7 macrophages with Concanavalin A (Con A; 25 µg/ml) and lipopolysaccaride (LPS; 1 µg/ml), respectively. Selective concentrations of glycoalkaloids and potato peel extracts were added simultaneously with Con A or LPS for 24h to investigate their potential to reduce inflammatory activity. KEY FINDINGS: α-Chaconine and solanidine significantly reduced interleukin-2 (IL-2) and interleukin-8 (IL-8) productions in Con A-induced Jurkat cells. The potato peel extracts did not influence cytokine production. In LPS-stimulated Raw macrophages, α-solanine, solanidine and two potato peel extracts significantly reduced induced NO production. SIGNIFICANCE: Our findings suggest that sub-cytotoxic concentrations of potato glycoalkaloids and potato peel extracts possess anti-inflammatory effects in vitro and with further investigation may be useful in the prevention of anti-inflammatory diseases.

A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery.

A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.

Activation of the STAT6 transcription factor in Jurkat T-cells by the herpesvirus saimiri Tip protein.

Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr(127) residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.

Attack the tumor counterattack-c-FLIP expression in Jurkat-T-cells protects against apoptosis induced by coculture with SW620 colorectal adenocarcinoma cells.

BACKGROUND: Cancer development relies on a variety of mechanisms that facilitate tumor growth despite the presence of a functioning immune system, employing different mechanisms to escape immune rejection. Tumors may eliminate tumor-infiltrating lymphocytes and suppress anti-tumor immune responses, a process called "tumor counterattack," based on activation-induced cell death via the FAS/FAS-ligand system. To overcome this tumor-cell survival strategy, we examined the hypothesis that the sensitivity of FAS mediated apoptosis of Jurkat-T-cells can be suppressed by FLIP transfection of Jurkat-T-cells. MATERIALS AND METHODS: Jurkat-T-cells were transfected with the FLICE-inhibitory protein FLIP in order to bestow them with a resistance to FAS-receptor-mediated apoptosis. FLIP-transfected and non-transfected Jurkat-T-cells were grown in coincubation with SW620 cells and the rates of apoptosis measured via FACS-analysis of Annexin-V. RESULTS: First, the tumor-counterattack described in the literature was confirmed. About 20% of Jurkat-T-Cells underwent apoptosis in coculture with SW620 cells. After coincubation of SW620 cells with FLIP transfected Jurkat-T-cells the apoptotic rate was significant reduced at levels below 4%. CONCLUSION: Transfection of Jurkat-T-cells with FLIP reduces the sensitivity of Jurkat-T-cells to FAS-mediated apoptosis and may lead to an improved capability to antagonize the inherent tumor survival strategy of SW620 cells.

Assessment of batch to batch variation in polyclonal antithymocyte globulin preparations.

BACKGROUND: Antithymocyte globulins (ATGs) are used to prevent and treat allograft rejection and graft versus host disease. They are purified IgG fractions derived from rabbits immunized with the Jurkat T-cell line (ATG-Fresenius) or thymus cells (Thymoglobulin). Differences not only in the amounts of leukocyte reactive antibodies but also in the antigens targeted by ATGs could potentially affect the clinical efficacy of different batches of these polyclonal antibody preparations. METHODS: Four batches of ATG-Fresenius and Thymoglobulin were compared regarding their capacity to interact with human leukocytes from healthy donors and kidney transplant recipients. Using flow cytometric assays, we analyzed the reactivity of these ATG preparations with Jurkat cells and with primary leukocytes. In addition, ATGs derived from different batches were probed with a panel of cell lines expressing high levels of ATG antigens. Their ability to mediate complement-mediated lysis of human monocytes and lymphocytes was also compared. RESULTS: Binding studies to leukocyte antigens and functional analysis pointed to a high conformity among different batches in both ATG preparations. CONCLUSIONS: From our in vitro data, it can be expected that ATGs derived from different batches will not differ in their clinical efficacy. Furthermore, the methods described in this study allow for a reliable analysis of ATG batches.

Essential role of the adaptor protein Nck1 in Jurkat T cell activation and function.

The non-catalytic region of tyrosine kinase (Nck) is proposed to play an essential role in T cell activation. However, evidence based on functional and biochemical studies has brought into question the critical function of Nck. Therefore, the aim of the present work was to investigate the role of Nck in T cell activation. To study this, the human Jurkat T cell line was used as a model for human T lymphocytes. The short interfering (si) RNA targeting Nck1 gene was used with electroporation to knock-down Nck1 protein expression in Jurkat T cells. Primary human CD4 T cells were also transfected with the siRNA of Nck1. The results showed that decreased Nck1 protein expression did not affect the apoptosis of the transfected Jurkat T cells compared with control siRNA-transfected cells and non-transfected cells. Upon CD3ε/CD28 stimulation, knock-down of Nck1 in Jurkat T cells caused a decrease in CD69 expression and in interleukin (IL)-2 secretion. Similarly, knock-down of Nck1 in primary CD4 T cells also caused decreased CD69 expression. However, no significant alterations of CD69 and IL-2 expression were found upon phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) stimulation. Knock-down of Nck1 had no effect on the proliferation of Jurkat T cells stimulated with either PHA or anti-T cell receptor (TCR) monoclonal antibody (C305). The reduced Nck1 expression in Jurkat cells was also associated with a reduced phosphorylation of extracellular regulated kinase (Erk)1 and Erk2 proteins upon CD3ε/CD28 stimulation. In conclusion, the decreased Nck1 protein in Jurkat T cells resulted in an impairment of TCR-CD3-mediated activation involving a defective Erk phosphorylation pathway.