Pyk2 is differentially regulated by β 1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

β₁ integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by β₁ integrin signaling in human T cells. Stimulation of Jurkat T cells with the α₄β₁ integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and α₄β₁ integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4⁺ T cells confirmed the ability of CD3-derived signals to synergize with β₁ integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell costimulation mediated specifically by β₁ integrins.     

CD59锚定蛋白对CD55介导的T细胞信号转导的增强效应

目的:研究糖基磷脂酰肌醇(GPI)锚定蛋白CD59对CD55介导T细胞信号转导的增强效应。方法:实验分为未转染的Jurkat细胞组(Ⅰ组)、转染空质粒的Jurkat细胞组(Ⅱ组)及转染CD59干扰质粒的Jurkat细胞组(Ⅲ组)。用RT-PCR检测3组细胞中的CD59基因表达水平。用噻唑蓝(MTT)比色法、免疫印迹技术和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对3组Jurkat细胞的增殖效应,Src家族酪氨酸激酶(SrcPTK)磷酸化的水平及细胞内钙离子的变化。结果:稳定转染后,Ⅲ组细胞CD59分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力,SrcPTK磷酸化水平及钙离子浓度均明显高于Ⅲ组(P<0.05);但Ⅰ组和Ⅱ组之间无差异。结论:CD59可增强CD55对T细胞信号转导的效应。     

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells

We demonstrate that pretreatment of primary CD4⁺, but not CD8⁺ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCRαβ). Specifically, anti-TCRαβ-mediated tyrosine phosphorylation of phospholipase C-γ1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca²⁺ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8⁺ T cells. Perturbation of TCRαβ signalling in CD4⁺ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCRαβ signalling, anti-CD3?-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCRαβ to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4⁺ and CD8⁺ T cells.     

Engagement of CD45 during in vitro priming enhances antigen-specific Th cell frequencies

CD45 is a transmembrane protein tyrosine phosphatase expressedby all lymphoid cells including T cells. Substantial experimentaldata has shown that CD45 maintains a permissive state for TCRsignaling. The highly glycosylated extracellular domain of CD45may be the site of Interaction with regulatory lectin-like counter-receptorson antigen-presenting cells. The mAb NDA5, recognizing a uniquebut broadly distributed epitope of CD45, was used to study thepossible immunoregulatory role of CD45 during antl-CD3 and antigen-specificCD4⁺ T cell activation. In vitro priming of peripheral bloodmononuclear cells with peptlde antigens in the presence of mAbNDA5 results in a higher frequency of antigen-specific T cells.The responses of both naive and memory T cells to peptide antigenswere sensitive to mAb NDA5-enhanced priming. Anti-CD3 activationof normal resting T cells, in the presence of mAb NDA5, resultedIn enhancement of tyrosine phosphorylation of specific intracellularproteins associated with TCR signal transduction. In cultureswithout antigen, mAb NDA5 down-regulated the cell surface expressionof both CD3 and CD4, yet did not stimulate proliferation ofresting T cells. Together these results suggest that engagementof CD45 during in vitro priming has a significant effect onthe development of antigen-specific T cell populations.     

CD59锚定蛋白对CD3介导Jurkat细胞活化信号转导中的增强效应

目的用前期构建好的CD59pSUPER-RNAi质粒转染Jurkat细胞,探讨CD59特异性沉默前后CD59对CD3介导Jurkat细胞活化信号转导的相关作用。方法采用Lipofectamine2000转染Jurkat细胞,经G418筛选建立稳定转染细胞系,利用RT-PCR检测转染后CD59在基因水平的表达抑制效果。实验分为未转染的Jurkat细胞组(A组),转染空质粒pSUPER组(B组)和转染CD59pSUPER-RNAi质粒的Jurkat细胞组(C组),用噻唑蓝(MTT)比色法,Western blot技术和激光共聚焦扫描显微镜分别检测交联CD59单抗与固相化CD3单抗联合作用对3组Jurkat细胞的增殖效应,ZAP-70酪氨酸磷酸化的水平及细胞浆内钙离子的变化。结果重组载体转染后,经G418筛选得到稳定转染细胞系,RT-PCR结果表明转染CD59pSUPER-RNAi质粒的Jurkat细胞组CD59分子的表达受到抑制。转染干扰质粒Jurkat细胞组CD59与CD3联合作用后细胞增殖能力,ZAP-70酪氨酸磷酸化水平及钙离子浓度均明显低于未转染组和转染空质粒组(P<0.05);但未转染组和转染空质粒组之间无差异。结论 CD59对CD3介导T细胞活化信号转导有增强效应。     

Age-related impairments in TCR/CD3 activation of ZAP-70 are associated with reduced tyrosine phosphorylations of zeta-chains and p59fyn/p56lck in human T cells

The expression and catalytic activity of the protein tyrosine kinase (PTK) ZAP-70 are needed for normal intracellular signaling through the T-cell receptor (TCR)/CD3 complex. However, the possible effect of aging on the catalytic activity of ZAP-70 in human peripheral blood T cells stimulated via the TCR/CD3 complex is unknown. The current studies show that T cells from a substantial proportion of elderly humans (12) exhibit significant reductions in the catalytic activity, but not expression of ZAP-70 when stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. In addition, the reduced catalytic activity of ZAP-70 in T cells from elderly subjects was not restored to the normal levels in response to ligation of CD4 receptors, suggesting defects in PTKs linked to both CD3 and CD4 receptors. Other experiments demonstrated that the age-related impairments of ZAP-70 activation in anti-CD3-stimulated T cells were accompanied by decreased tyrosine phosphorylations of zeta-chains and autophosphorylations of the PTKs p561ck/p59fyn. Moreover, the age-related defects in these early TCR/CD3-mediated phosphorylation events were readily detectable in both CD45RO+ memory and CD45RA+ naive T cells. Thus, these results suggest that defects in early TCR/CD3-mediated phosphorylation events among CD45RO+ memory and CD45RA+ naive T cells from certain elderly humans may con tribute to impaired induction of ZAP-70 catalytic activity.     

Tyrosine phosphorylation in peripheral T cells of kidney transplant recipients: analyses of baseline levels and response to T cell receptor stimulation.

Impaired immunosurveillance in recipients of organ transplants has been attributed to alleviation of T cell functions. We analyzed the phosphorylation of tyrosine residues in T cells of peripheral blood, and after T cell receptor (TCR) stimulation. The TCR was stimulated by OKT3 monoclonal antibody (mAb) in non-separated heparinized blood specimens of patients (n=64) and healthy controls (n=25). After fixation and red cell lysis, lymphocytes were permeabilized by saponin. Subsequently, intracellular phosphotyrosine residues and surface CD3 antigen were stained simultaneously with specific mAbs. We analyzed transplant recipients and healthy donors for baseline levels of total cellular tyrosine phosphorylation and for increase in phosphotyrosine content following stimulation by OKT3. Phosphotyrosine levels were significantly lower in non-stimulated T cells of kidney transplant recipients compared to controls (p=0.004). There was a marked variability in the levels of tyrosine phosphorylation among transplanted patients (p=0.02). T cell receptor stimulation by OKT3 mAb in vitro led to a strong increase of tyrosine phosphorylation in all specimens of patients and healthy controls. In conclusion, we demonstrated decreased phosphotyrosine levels in T cells of kidney transplant recipients compared to healthy donors. However, increase in tyrosine phosphorylation was not impaired in all patients as a result of TCR stimulation.     

The CD45 protein tyrosine phosphatase is required for the completion of the activation program leading to lymphokine production in the Jurkat human T cell line.

Stimulation of the T cell antigen receptor, TCR-CD3, induces tyrosine phosphorylation of specific cellular proteins through activation of a tyrosine kinase. The possible regulatory role of the CD45 protein tyrosine phosphatase in this process was explored by studying the functional properties of cellular variants of the Jurkat T cell line which have been selected to have normal levels of the TCR-CD3 complex, but low or negative expression of CD45. These variants had less than 20% of the normal membrane tyrosine phosphatase activity. Triggering the TCR-CD3 receptor on the CD45 variants with anti-CD3 mAb induced the activation of a tyrosine kinase. Tyrosine phosphorylation of cellular substrates as well as of the CD3 zeta chain was qualitatively comparable to normal cells although the extent of stimulation was lower. No differences were observed between the variants and the normal cells in the duration of the tyrosine phosphorylation signal. The increase in intracellular calcium concentration following receptor stimulation was also less efficient, suggesting that CD45 is necessary for optimal generation of the second messengers of the activation. The CD45 deficient cells secreted highly reduced levels of lymphokines (IL-2, IL-3 or GM-CSF) after activation by anti-CD3 mAb combined with the phorbol ester TPA. This impaired lymphokines production is related to the absence of CD45 since a CD45+ revertant subclone, isolated from one CD45- clone, produced normal levels of cytokines upon activation via CD3, while CD45- subclones were unable to secrete cytokines following activation via CD3. However, upon activation with Ca2+ ionophore and PMA, all CD45- (sub)clones secreted cytokines at levels comparable to those produced by CD45+ cells. These results show that CD45 is required for cytokine production after activation via the TCR-CD3 complex.     

Selective coupling of the T cell antigen receptor to phosphoinositide-derived diacylglycerol production in HPB-ALL T cells correlates with CD45-regulated p59fyn activity

In HPB-ALL T-cells the p59^fyn tyrosine kinase is regulated by the CD45 phosphotyrosine phosphatase and plays a critical role in coupling the T cell receptor (TCR) to the generation of intracellular signals which include diacylglycerol (DAG) production and protein kinase C activation. The aim of this study was to determine the phospholipid pools from which the DAG is generated and to identify which phospholipase activities are regulated by the TCR. When CD45⁺cells were pre-labeled with [³H] arachidonic acid, CD3-antigen cross-linking stimulated negligible increases in both [³H]DAG and [³H] phosphatidic acid (PA). However, CD3 monoclonal antibody (mAb) induced an increase of 300 % in [³H]PA when the cells were permeabilized with streptolysin-O, and this correlated with increased levels of protein tyrosine phosphorylation. Stimulation of [³H] PA production upon CD3 cross-linking was 77 % lower in permeabilized CD45^? cells than in CD45⁺ cells, consistent with the reduced activity of p59^fyn in CD45^? cells. The stimulated production of PA was not mediated by activation of phospholipase D (PLD), although the presence of a G-protein-regulated PLD activity was established. The CD3-induced increase in total inositol phosphates (InsP) in permeabilized cells was similar to the stimulated production of [³H] PA production in both CD45⁺ and CD45^? cells. Dose-response curves for InsP and PA production triggered by CD3 mAb were super-imposable and the production of InsP and PA over a range of Ca²⁺ concentrations was comparable. Differential labeling of phospholipids with ³H-labeled fatty acids revealed that CD3-induced PA production reflected incorporation of label into the phosphatidylinositol pool. Our data suggest that in HPB-ALL cells the production of DAG following CD3-antigen cross-linking can be fully accounted for by the selective coupling of the TCR to breakdown of phosphatidylinositol-(4,5)-bisphosphate as the result of phospholipase Cγ₁ activation. This event correlates with the activity of the CD45-regulated TCR-associated tyrosine kinase, p59^fyn.     

HIV-1 glycoprotein gp120 disrupts CD4-p56lck/CD3-T cell receptor interactions and inhibits CD3 signaling

Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV-1 glycoprotein gp120 inhibited CD3-induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4-linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3-T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti-CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4-associated p56^lck and CD4-linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti-CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3-induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide-specific phospholipase C-γ-1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3-induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4-p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down-modulation of CD4-p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3-linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.     

WSX-1 over-expression in CD4(+) T cells leads to hyperproliferation and cytokine hyperproduction in response to TCR stimulation

WSX-1 is a component of the IL-27R. Analyses of WSX-1 knockout (WSX-1(-/-)) mice have shown that IL-27/WSX-1 signaling is essential for the proper development of T(h)1 responses and that WSX-1 can suppress cellular activation and pro-inflammatory cytokine production. We have generated transgenic mouse lines over-expressing the WSX-1 gene under the control of the T cell-specific CD2 promoter (WSX-1 Tg mice). Unexpectedly, like activated CD4(+) T cells from WSX-1(-/-) mice, activated CD4(+) T cells from WSX-1 Tg mice showed increased proliferation, augmented IL-2 production and up-regulated surface expression of activation markers. IL-27-mediated tyrosine phosphorylation of STAT1 was also enhanced in WSX-1 Tg CD4(+) T cells, but STAT3 activation was normal. Exogenous IL-27 supported the proliferation of wild-type CD4(+) T cells but suppressed that of WSX-1 Tg cells. WSX-1 over-expression increased IFN-gamma production in T(h)1-polarized CD4(+) T cells, but also promoted T(h)2 cytokine production under T(h)1-polarizing conditions. Importantly, WSX-1 over-expression failed to suppress T(h)2 cytokine production under T(h)2-polarizing conditions. Cytokine hyperproduction was also observed in vivo in WSX-1 Tg mice injected with Con A. Our data suggest that WSX-1 plays a pivotal role in regulating T cell responsiveness to TCR stimulation and that the correct balance of STAT1/STAT3 activation downstream of IL-27R engagement is crucial for the physiological function of IL-27.     

T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.     

Inhibition of TCR/CD3-mediated signaling by a mutant of the hematopoietically expressed G16 GTP-binding protein

We have investigated the role of the hematopoietically expressed G16 GTP-binding protein on T cell activation. We constructed transfectants of Jurkat T cells that express a function-deficient mutant of Gα16 predicted to prevent activation of this G protein. Upon stimulation with anti-CD3ϵ antibodies, mutant Gα16 transfectants display a profound defect in the production of IL-2 and IL-10, as well as in the expression of CD69. In contrast, the phorbol 12-myristate 13-acetate (PMA)-induced IL-10 production and CD69 expression, and the ionomycin plus PMA-induced IL-2 production are not affected. Consistent with the reduction in cytokine production is the inhibition of early signaling events in the mutant Gα16-expressing cells. There are significant reductions in anti-ϵ-induced tyrosine phosphorylation of ζ, ϵ, ZAP-70, and phospholipase Cγ1, as well as in intracellular Ca²⁺ mobilization. In accordance with the effects on tyrosine phosphorylation is the reduction of TCR/CD3-mediated Fyn and Lck activities in Gα16 mutant cells. Even though the mechanism through which the Gα16 mutant mediates inhibition of T cell activation is not known, the data suggest a model where G proteins become activated upon TCR/CD3 engagement and regulate the activation of tyrosine kinases and subsequent downstream signaling events that lead to the activation of cytokine genes.     

Signalling initiated with CD4–TCR or TCR–TCR interactions: comparison of tyrosine phosphorylation patterns and CD45 effects

Antigen-triggered response in T cells is mediated by the T cell receptor (TCR)/CD3-complex. This signalling, however, is modulated by a number of other surface molecules. Among the most important of these is the CD4/CD8 molecule which associates with the TCR/CD3-complex and binds to the MHC complex. The molecular mechanisms involved in interactions between TCR–TCR and TCR–CD4 are not fully understood. We have earlier described an experimental model that allows us to dissect signals involving CD4–TCR interactions and those involving TCR–TCR interactions using a mouse CD4⁻CD8⁻ T cell hybridoma cell-line transfected either with the TCR from a mouse T-helper ₂ cell-line (D10) alone or with both the TCR and the CD4 molecule. To further characterize these two different modes of signalling in T lymphocytes we have studied the tyrosine phosphorylation patterns resulting from these interactions. In addition, we have studied the modulatory effect of the CD45 molecule on these interactions. In contrast to some earlier reports, we found that both the patterns of induced tyrosine phosphorylation and the effects of CD45 modulation were essentially similar in the CD4–TCR and the TCR–TCR signal transduction cascades. The results are consistent with a purely synergistically amplifying function for CD4 on the TCR-mediated signalling.     

Differential T cell receptor-mediated signaling in naive and memory CD4 T cells

Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3ζ subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3ζ in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72^syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.     

CD2 singnaling in T cells involves tyrosine phosphorylation and activation of the Tec family kinase, EMT/ITK/TSK

Ligation of the CD2 cell surface glycoprotein expressed on Tlymphocytes and NK cells induces protein tyrosine phosphorylationand activation of the Src kinases, LCK and FYN. We show herethat in Jurkat T leukemia cells and in peripheral blood T cells,CD2 stimulation also leads to tyrosine phosphorylation and activationof the Tec family kinase, EMThTKITSK. Activation of EMT by CD2was induced by mitogenic pairs of CD2 mAb, certain single CD2mAb followed by secondary antibody cross-linking, and CD58-bearingsheep red blood cells. With the use of different Jurkat cellmutants it was demonstrated that CD2-mediated activation ofEMT required expression of LCK, but did not require surfaceexpression of the CD3 chain. Receptor-mediated activation ofLCK does not in itself lead to activation of this Tec kinasesince induction of LCK by ligation of CD4 or CD5 did not resultin activation of EMT. The activation of EMT during CD2 signalingsuggests an important role for this kinase in CD2 co-stimulationof T cell responses.     

CD28 affects the earliest signaling events generated by TCR engagement

The efficiency and magnitude of T cell responses are influenced by ligation of the co-stimulatory receptor CD28 by B7 molecules expressed on antigen-presenting cells (APC). In contrast to most previous studies in which agonistic anti-TCR/CD3 and anti-CD28 antibodies were employed, here we have investigated the contribution of CD28 to T cell activation under physiological conditions of antigen presentation. Jurkat T cells and primary T cells from TCR-transgenic mice stimulated with superantigen and antigen, respectively, presented by B7-expressing APC were utilized. In both systems we show that inhibiting CD28/B7 interaction resulted in impaired TCR-induced tyrosine phosphorylation of the signal-transducing ζ chain and ZAP-70. Consistent with a blockade of TCR-proximal signaling events, Jurkat cells stimulated in the absence of CD28 ligation were found to have strongly diminished tyrosine phosphorylation of cellular substrates and downstream signaling pathways such as Ca²⁺ /calcineurin, ERK/MAPK and JNK. Our results provide evidence for a role of CD28 in enhancing TCR signaling capacity during the earliest stages of T cell : APC interaction.