To T Cell Receptor Stimulation Research Articles

Cis interaction of CD153 with TCR/CD3 is crucial for the pathogenic activation of senescence-associated Tcells.

With age, senescence-associated (SA) CD4+ Tcells that are refractory to Tcell receptor (TCR) stimulation are increased along with spontaneous germinal center (Spt-GC) development prone to autoantibody production. We demonstrate that CD153 and its receptor CD30 are expressed in SA-T and Spt-GC B cells, respectively, and deficiency of either CD153 or CD30 results in the compromised increase of both cell types. CD153 engagement on SA-T cells upon TCR stimulation causes association of CD153 with the TCR/CD3 complex and restores TCR signaling, whereas CD30 engagement on GC B cells induces their expansion. Administration of an anti-CD153 antibody blocking the interaction with CD30 suppresses the increase in both SA-T and Spt-GC B cells with age and ameliorates lupus in lupus-prone mice. These results suggest that the molecular interaction of CD153 and CD30 plays a central role in the reciprocal activation of SA-T and Spt-GC B cells, leading to immunosenescent phenotypes and autoimmunity.

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

Preclinical characterization and efficacy of 4R9, a novel immune checkpoint blockade targeting CEACAM1 for cancer therapy.

e14155 Background: CEACAM1 is one of the several immune checkpoint receptors expressed on T cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction induces downregulation of ZAP70 phosphorylation in response to T cell receptor (TCR) stimulation. Also, CEACAM1 is highly expressed on non-small cell lung cancer (NSCLC) and its expression correlated with cancer progression and poor prognosis. We developed a fully human monoclonal antibody 4R9, human CEACAM1-targeting antibody. Methods: T cell activation of 4R9 was determined by NFAT-luciferase reporter assay with CEACAM1 overexpressing Jurkat stable cells. In vitro efficacy of 4R9 was examined using NK-mediated tumor cell killing assay. The anti-tumor efficacy of 4R9 alone or in combination was studied in vivo in a humanized mouse model engrafted with NSCLC patient-derived tumor xenografts. Results: Anti-CEACAM1 antibody 4R9 binds to CEACAM1 overexpressed in HEK293 or Jurkat cells but not to other CEA family members. 4R9 blocks CEACAM1-CEACAM1 homophilic interaction by binding to N domain of CEACAM1. CEACAM1-CEACAM1 homophilic interaction induced downregulation of ZAP70 phosphorylation in response to TCR stimulation in CEACAM1 overexpressing Jurkat stable cell line, which was rescued by 4R9 resulting in augmentation of NFAT activity and IL-2 expression. NK cell-mediated tumor lysis was increased by 4R9 in a CEACAM1 expression-dependent manner. Out of 49 NSCLC tumor tissues, 20 cases exhibited dominant expression of CEACAM1 over PD-L1 with 6 cases showing > 50% of CEACAM1 positivity. In a single mouse trial using NSCLC PDX-huNSG mouse model, 4R9 (20 mpk, 2qW) suppressed tumor progression more than 30% as monotherapy (10/19) as well as in combination (16/22) with pembrolizumab (5 mpk, 2qW). Moreover, PDX of adenocarcinoma origin with more than 50% of CEACAM1 expression were more efficiently prohibited for progression with 4R9, suggesting potential therapeutic use of 4R9 in patients with NSCLC. Conclusions: Anti-CEACAM1 antibody blocked CEACAM1-mediated negative regulation and restored T/NK cell activities. Different expression patterns of CEACAM1 compared with PD-L1 and efficacies in a single mouse trial support the rationale for developing anti-CEAEAM1 antibody as cancer therapeutics.

Special AT-rich sequence binding protein 1 is required for maintenance of T cell receptor responsiveness and development of experimental autoimmune encephalomyelitis.

ABSTRACTThe genome organizer special AT‐rich sequence binding protein 1 (SATB1) regulates specific functions through chromatin remodeling in T helper cells. It was recently reported by our team that T cells from SATB1 conditional knockout (SATB1cKO) mice, in which the Satb1 gene is deleted from hematopoietic cells, impair phosphorylation of signaling molecules in response to T cell receptor (TCR) crosslinking. However, in vivo T cell responses upon antigen presentation in the absence of SATB1 remain unclear. In the current study, it was shown that SATB1 modulates T cell antigen responses during the induction and effector phases. Expression of SATB1 was upregulated in response to TCR stimulation, suggesting that SATB1 is important for this antigen response. The role of SATB1 in TCR responses and induced experimental autoimmune encephalomyelitis (EAE) was therefore examined using the myelin oligodendrocyte glycoprotein peptide 35‐55 (MOG35‐55) and pertussis toxin. SATB1cKO mice were found to be resistant to EAE and had defects in IL‐17‐ and IFN‐γ‐producing pathogenic T cells. Thus, SATB1 expression appears necessary for T cell function in the induction phase. To examine SATB1 function during the effector phase, a tamoxifen‐inducible SATB1 deletion system, SATB1cKO‐ER‐Cre mice, was used. Encephalitogenic T cells from MOG35‐55‐immunized SATB1cKO‐ER‐Cre mice were transferred into healthy mice. Mice that received tamoxifen before the onset of paralysis were resistant to EAE. Furthermore, no disease progression occurred in recipient mice treated with tamoxifen after the onset of EAE. Thus, SATB1 is essential for maintaining TCR responsiveness during the induction and effector phases and may provide a novel therapeutic target for T cell‐mediated autoimmune diseases.

Activation-induced FOXP3 isoform profile in peripheral CD4+ T cells is associated with coronary artery disease

Background and aimsThe expression of FOXP3 isoforms affects regulatory T (Treg) cell function. Reduced Treg cell function has been associated with coronary artery disease (CAD). However, alternative splicing of FOXP3 in CAD has not been investigated. MethodsFOXP3 splice variants and IL17A transcripts in peripheral blood mononuclear cells from stable CAD patients and healthy controls were quantified, and FOXP3 isoform expression in response to T cell receptor (TCR) stimulation or LDL was analyzed by flow cytometry. ResultsCompared to healthy controls, CAD patients expressed significantly more FOXP3 transcripts that included exon 2, whereas alternative splicing of exon 7 in correlation with IL17A expression was reduced. Moreover, TCR stimulation, as well as exposure to LDL, decreased alternative splicing of FOXP3 in CD4+ T cells in vitro. ConclusionsOur results demonstrate that blood mononuclear cells in stable CAD patients express a ratio of FOXP3 isoforms that is characteristic for activated CD4+ T cells.

Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection.

Systemic ATP Levels Suppress the Function of CD4+ T Cells in Sepsis by Impairing Autocrine Purinergic Signaling

T cell suppression in critical care patients can cause sepsis. The underlying mechanisms leading to T cell suppression are largely unknown. We have previously demonstrated that T cell activation requires ATP release and autocrine purinergic signaling via P2X receptors. Here we show that systemic ATP released in response to injury and inflammation interferes with these autocrine signaling processes resulting in suppression of CD4+ T cell activation. Using Fluo-4 and flow cytometry, we found that cytosolic Ca2+ signaling in response to T cell receptor (TCR)/CD28 stimulation of CD4+ T cells from sepsis patients was significantly lower compared to healthy controls (Fig. 1A). Plasma ATP levels were ~3 times higher in sepsis patients (101 ± 9 nM) than in healthy controls (31 ± 7 nM; p<0.001). Adding ATP or the non-hydrolyzable ATP analog ATPγS time- and dose-dependently impaired TCR stimulation induced Ca2+ signaling of healthy CD4+ T cells (Fig. 1B; *p<0.05 vs. untreated) and suppressed proliferation of mouse splenocytes by up to 80%. Taken together these findings suggest that elevated systemic ATP in the plasma of critical care patients suppresses the function of T cells by interfering with their autocrine purinergic signaling. Targeting this inhibitory process may improve T cell function and outcome after sepsis. The study was supported by NIH grants R01 GM51477, R01 GM60475, R01 AI080582, T32 GM103702, and DFG grant LE-3209/1-1.

'Default' generated neonatal regulatory T cells are hypomethylated at conserved non-coding sequence 2 and promote long-term cardiac allograft survival.

Regulatory T (Treg) cells play an important role in the maintenance of immune self-tolerance and homeostasis. We previously reported that neonatal CD4(+) T cells have an intrinsic 'default' mechanism to become Treg (neoTreg) cells in response to T-cell receptor (TCR) stimulation. However, the underlying mechanisms are unclear and the effects of neoTreg cells on regulating immune responses remain unknown. Due to their involvement in Foxp3 regulation, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b during the induction of neoTreg cells in the Foxp3(gfp) mice. The function of neoTreg cells was assessed in an acute allograft rejection model established in RAG2(-/-) mice with allograft cardiac transplantation and transferred with syngeneic CD4(+) effector T cells. Following ex vivo TCR stimulation, the DNMT activity was increased threefold in adult CD4(+) T cells, but not significantly increased in neonatal cells. However, adoptively transferred neoTreg cells significantly prolonged cardiac allograft survival (mean survival time 47 days, P < 0.001) and maintained Foxp3 expression similar to natural Treg cells. The neoTreg cells were hypomethylated at the conserved non-coding DNA sequence 2 locus of Foxp3 compared with adult Treg cells. The DNMT antagonist 5-aza-2'-deoxycytidine (5-Aza) induced increased Foxp3 expression in mature CD4(+) T cells. 5-Aza-inducible Treg cells combined with continuous 5-Aza treatment prolonged graft survival. These results indicate that the 'default' pathway of neoTreg cell differentiation is associated with reduced DNMT1 and DNMT3b response to TCR stimulus. The neoTreg cells may be a strategy to alleviate acute allograft rejection.

Participation of the Cell Polarity Protein PALS1 to T-Cell Receptor-Mediated NF-κB Activation

BackgroundBeside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation.Methodology/Principal FindingsBy combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB.ConclusionsThe cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

Signaling thresholds govern heterogeneity in IL‐7‐receptor‐mediated responses of naïve CD8+ T cells

Variable sensitivity to T-cell-receptor (TCR)- and IL-7-receptor (IL-7R)-mediated homeostatic signals among naïve T cells has thus far been largely attributed to differences in TCR specificity. We show here that even when withdrawn from self-peptide-induced TCR stimulation, CD8+ T cells exhibit heterogeneous responses to interleukin-7 (IL-7) that are mechanistically associated with IL-7R expression differences that correlate with relative CD5 expression. Whereas CD5hi and CD5lo T cells survive equivalently in the presence of saturating IL-7 levels in vitro, CD5hi T cells proliferate more robustly. Conversely, CD5lo T cells exhibit prolonged survival when withdrawn from homeostatic stimuli. Through quantitative experimental analysis of signaling downstream of IL-7R, we find that the enhanced IL-7 responsiveness of CD5hi T cells is directly related to their greater surface IL-7R expression. Further, we identify a quantitative threshold in IL-7R-mediated signaling capacity required for proliferation that lies well above an analogous threshold requirement for survival. These distinct thresholds allow subtle differences in IL-7R expression between CD5lo and CD5hi T cells to give rise to significant variations in their respective IL-7-induced proliferation, without altering survival. Heterogeneous IL-7 responsiveness is observed similarly in vivo, with CD5hi naïve T cells proliferating preferentially in lymphopenic mice or lymphoreplete mice administered with exogenous IL-7. However, IL-7 in lymphoreplete mice appears to be maintained at an effective level for preserving homeostasis, such that neither CD5hi IL-7Rhi nor CD5lo IL-7Rlo T cells proliferate or survive preferentially. Our findings indicate that IL-7R-mediated signaling not only maintains the size but also impacts the diversity of the naïve T-cell repertoire.

Novel function of perforin in negatively regulating CD4+ T cell activation by affecting calcium signaling

Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic T lymphocytes (CTLs) and natural killer cells. However, whether it also plays a role in conventional CD4(+) T cell function remains unclear. Here we report that in perforin-deficient (PKO) mice, CD4(+) T cells are hyperproliferative in response to T cell receptor (TCR) stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD4(+) T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFAT1. Our results indicate that perforin plays a negative role in regulating CD4(+) T cell activation and immune response by affecting TCR-dependent Ca(2+) signaling.

Negative regulation of T cell receptor signaling by NFkappaB2/p100 (87.4)

Abstract The positive regulation of the NFκB signaling pathway in response to T cell receptor (TCR) stimulation has been well studied. However, little is known about the negative regulation of this pathway in T cells. This negative regulation is crucial in controlling the duration of TCR signaling and preventing abnormal lymphocyte activation and proliferation. Therefore, understanding the negative regulation of TCR-mediated NFκB signaling is essential in understanding the mechanisms involved in T cell function and homeostasis. TCR stimulation of human CD4+ T cells resulted in an increase in NFκB2/p100 expression with minimal increase in p52, its cleavage product. Due to the presence of inhibitory ankyrin-repeats in the unprocessed p100, this observation suggests that p100 may function as a negative regulator of the NFκB pathway. Consistent with this hypothesis, ectopic expression of p100 inhibited TCR-mediated NFκB activity and IL-2 production in Jurkat T cells. Conversely, knockdown of p100 expression enhanced NFκB transcriptional activity and IL-2 production upon TCR activation. p100 inhibited the pathway by binding and sequestering Rel transcription factors in the cytoplasm without affecting the activity of the upstream IκB kinase (IKK). Taken together, this study indicates that NFκB2/p100 can act as an inhibitory molecule in the TCR-mediated NFκB signaling pathway. This work was supported by a grant from the National Institutes of Health to A.T.T.

TEC FAMILY KINASES IN T LYMPHOCYTE DEVELOPMENT AND FUNCTION

The Tec family tyrosine kinases are now recognized as important mediators of antigen receptor signaling in lymphocytes. Three members of this family, Itk, Rlk, and Tec, are expressed in T cells and activated in response to T cell receptor (TCR) engagement. Although initial studies demonstrated a role for these proteins in TCR-mediated activation of phospholipase C-gamma, recent data indicate that Tec family kinases also regulate actin cytoskeletal reorganization and cellular adhesion following TCR stimulation. In addition, Tec family kinases are activated downstream of G protein-coupled chemokine receptors, where they play parallel roles in the regulation of Rho GTPases, cell polarization, adhesion, and migration. In all these systems, however, Tec family kinases are not essential signaling components, but instead function to modulate or amplify signaling pathways. Although they quantitatively reduce proximal signaling, mutations that eliminate Tec family kinases in T cells nonetheless qualitatively alter T cell development and differentiation.

The Yins of T Cell Activation

Transcription factors activated in response to T cell receptor (TCR) signaling include nuclear factor of activated T cells (NFAT) family, which is highly phosphorylated and thereby maintained in the cytoplasm of resting T cells, the nuclear factor NF-kappaB, which is kept in the cytoplasm of resting cells through its association with the inhibitor protein IkappaB, and activating protein-1 (AP-1), which is only transcribed after TCR stimulation. Negative regulators of TCR signaling can be divided into two groups: Class 1 regulators help maintain the quiescent state of unstimulated T cells, whereas class 2 regulators are themselves transcriptionally induced in response to TCR signaling and serve to limit and terminate the activating signal. Class 1 regulators include the autoinhibitory domain of the phosphatase calcineurin; IkappaB and its transcriptional activators Foxj1 and Foxo3a; and various transcriptional coregulators that inhibit interleukin-2 (IL-2) production. Class 2 regulators include the calcipressins, which, like NFATp and NFAT4 are feedback inhibitors of calcineurin-NFAT signaling, IkappaB, and the mitogen-activated protein kinase (MAPK) phosphatases, which inhibit MAPK signaling and thus the nuclear localization of AP-1 components.

Intracellular calcium release is required for caspase-3 and -9 activation.

Increase in intracellular Ca2+ [Ca2+]i regulates many biological functions including apoptosis, but the protein(s) linking [Ca2+]i and apoptosis are not completely understood. We have previously shown that IP3R-deficient cells are resistant to T-cell receptor (TCR)-induced apoptosis due to lack of Ca2+ release from endoplasmic reticulum (ER) and calcineurin activation. Here we show that caspase-9 and -3 are not activated in IP3R-deficient cells after TCR stimulation, consistent with the resistance of these cells to apoptosis. However, we also demonstrate that Bcl-2 expression in IP3R-deficient cells is comparable to control cells. Taken together, these results strongly suggest that IP3R-mediated Ca2+ release plays a critical role in regulating the activity of caspases-3 and -9 independent of Bcl-2.

Functional correction of T cells derived from patients with the Wiskott-Aldrich syndrome (WAS) by transduction with an oncoretroviral vector encoding the WAS protein.

T-cell dysfunction is thought to be central to the immunodeficiency state seen in patients with the Wiskott-Aldrich syndrome (WAS). Aspects of the WAS phenotype have been corrected in other cell types on introduction of the normal WAS protein (WASP), but the potential for correction of the T-cell defects has not been evaluated. Here we demonstrate that an oncoretroviral vector encoding WASP and green fluorescent protein (GFP), and pseudotyped with the RD114 envelope protein, efficiently transduces primary human T cells derived from WAS patients. Transcription initiated at the oncoretroviral long terminal repeat (LTR) results in levels of WASP that, while lower than those seen in normal control T cells, resulted in correction of the deficient proliferative response to T-cell receptor (TCR) stimulation characteristic of WAS. IL2 secretion after TCR stimulation was partially corrected. Control primary T cells transduced with the same vector responded normally to TCR stimulation, and showed no increase in WASP expression. The demonstration that correction of T cell defects can be achieved by gene transfer supports continued efforts to develop gene therapy for WAS.

A Role for Wiskott-Aldrich Syndrome Protein in T-cell Receptor-mediated Transcriptional Activation Independent of Actin Polymerization

Wiskott-Aldrich syndrome protein (WASP) plays a key role in cytoskeletal rearrangement and transcriptional activation in T-cells. Recent evidence links WASP and related proteins to actin polymerization by the Arp2/3 complex. To study whether the role of WASP in actin polymerization is coupled to T-cell receptor (TCR)-mediated transcriptional activation, we made a series of WASP deletion mutants and tested them for actin co-localization, actin polymerization, and transcriptional activation of NFAT. A WASP mutant with a deletion in the C-terminal region (WASPDeltaC) that is defective in actin polymerization potentiated NFAT transcription following TCR activation by anti-CD3 and anti-CD3/CD28 antibodies, but not by phorbol 12-myristate 13-acetate/ionomycin. Furthermore, cotransfection of a dominant-active mutant (WASP-WH2-C) for Arp2/3 polymerization did not inhibit NFAT activation. Finally, by analyzing a series of WASP double-domain deletion mutants, we determined that the WASP homology-1 domain is responsible for NFAT transcriptional activation. Our results suggest that WASP activates transcription following TCR stimulation in a manner that is independent of its role in Arp2/3-directed actin polymerization.

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

CD8 coreceptor extinction in signaled CD4(+)CD8(+) thymocytes: coordinate roles for both transcriptional and posttranscriptional regulatory mechanisms in developing thymocytes.

T-cell development in the thymus is characterized by changing expression patterns of CD4 and CD8 coreceptor molecules and by changes in CD4 and CD8 gene transcription. In response to T-cell receptor (TCR) signals, thymocytes progress through developmental transitions, such as conversion of CD4(+)CD8(+) (double-positive [DP]) thymocytes into intermediate CD4(+)CD8(-) thymocytes, that appear to require more-rapid changes in coreceptor expression than can be accomplished by transcriptional regulation alone. Consequently, we considered the possibility that TCR stimulation of DP thymocytes not only affects coreceptor gene transcription but also affects coreceptor RNA stability. Indeed, we found that TCR signals in DP thymocytes rapidly destabilized preexisting CD4 and CD8 coreceptor RNAs, resulting in their rapid elimination. Destabilization of coreceptor RNA was shown for CD8alpha to be dependent on target sequences in the noncoding region of the RNA. TCR signals also differentially affected coreceptor gene transcription in DP thymocytes, terminating CD8alpha gene transcription but only transiently reducing CD4 gene transcription. Thus, posttranscriptional and transcriptional regulatory mechanisms act coordinately in signaled DP thymocytes to promote the rapid conversion of these cells into intermediate CD4(+)CD8(-) thymocytes. We suggest that destabilization of preexisting coreceptor RNAs is a mechanism by which coreceptor expression in developing thymocytes is rapidly altered at critical points in the differentiation of these cells.

Increase in the specific activity of p50 csk in proliferating T cells correlates with decreased specific activity of p56 lck and p59 fyn and reduced phosphorylation of CD3 subunits

Depending on their prior antigen recognition history, mature T cells respond with different functional outcomes to T cell receptor (TCR) stimulation. These functional outcomes include proliferation, anergy and cell death. The biochemical basis underlying differential responses by mature T cells at different stages of their developmental pathway to TCR stimulation remains to be determined. We have previously shown that proliferating but not naive T cells were susceptible to apoptosis after TCR stimulation and that the tyrosine phosphorylation of TCRζ, CD3γ, and CD3ϵ in proliferating T cells was decreased after TCR stimulation. In this study, we determined whether differences in phosphorylation between naive and proliferating T cells were due to altered regulation of p56 lck (Lck) or p59 fyn (Fyn) by their positive or negative regulators, CD45 or p50 csk (Csk), respectively. We found that Lck was expressed at the same level and had the same phosphotyrosine content in naive and proliferating T cells. However, its autophosphorylation activity was lower in proliferating cells, corresponding to a 2-fold decrease in its specific kinase activity. Similarly, the specific kinase activity of Fyn was also decreased by about 2-fold in proliferating T cells. In contrast, although Csk was expressed at the same level in both cell types its specific kinase activity was increased by 6-fold in proliferating T cells. The tyrosine phosphatase CD45, a positive regulator of src-family kinases, was overexpressed by 3- to 6-fold in proliferating cells. However, the specific activity of CD45 in naive and proliferating T cells was the same. Therefore, although the protein expression level of CD45 was increased in proliferating T cells it only partially compensated for the hyperactivity of Csk resulting in a 2-fold reduction in the specific activity of Lck and Fyn in proliferating T cells.