TCR engagement increases hypoxia-inducible factor-1 alpha protein synthesis via rapamycin-sensitive pathway under hypoxic conditions in human peripheral T cells

Peripheral T cells encounter rapid decrease in oxygen tension because they are activated by Ag recognition and migrate into inflammatory sites or tumors. Activated T cells, therefore, are thought to have such machineries that enable them to adapt to hypoxic conditions and execute immune regulation in situ. We have recently shown that survival of CD3-engaged human peripheral blood T cells is prolonged under hypoxic conditions and hypoxia-inducible factor-1 (HIF-1) and its target gene product adrenomedullin play a critical role for the process. It is also shown that hypoxia alone is not sufficient, but TCR-mediated signal is required for accumulation of HIF-1alpha in human peripheral T cells. In the present study, we showed that TCR engagement does not influence hypoxia-dependent stabilization but stimulates protein synthesis of HIF-1alpha, most possibly via PI3K/mammalian target of rapamycin system, and that expression of HIF-1alpha and its target genes is blocked by treatment with rapamycin. Since some of those gene products, e.g., glucose transporters and phosphoglycerokinase, are considered to be essential for glycolysis and energy production under hypoxic conditions and adequate immune reaction in T cells, this TCR-mediated synthesis of HIF-1alpha may play a pivotal role in peripheral immune response. Taken together, our results may highlight a novel aspect of downstream signal from Ag recognition by TCR and a unique pharmacological role of rapamycin as well.     

Differential responses of invariant V alpha 24J alpha Q T cells and MHC class II-restricted CD4+ T cells to dexamethasone.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.     

SLP-76 binding to p56lck: a role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex.

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185-194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.     

Crosstalk of hypoxia-mediated signaling pathways in upregulating plasminogen activator inhibitor-1 expression in keloid fibroblasts

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor-1 (PAI-1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia-mediated PAI-1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia-induced upregulation of PAI-1 expression is mainly controlled by hypoxia inducible factors-1alpha (HIF-1alpha) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2). Treatment of cells with PI-3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia-induced PAI-1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia-induced stabilization of HIF-1alpha protein, attenuation of the steady-state level of HIF-1alpha mRNA, and its DNA-binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia-induced PAI-1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia-mediated stabilization of HIF-1alpha protein and the steady-state level of HIF-1alpha mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3-K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia-mediated induction of PAI-1 expression via activation of HIF-1alpha in keloid fibroblasts.     

Proteasome inhibitors block Ras/ERK signaling pathway resulting in the downregulation of Fas ligand expression during activation-induced cell death in T cells

Activation-induced cell death (AICD) plays a critical role in the maintenance of homeostasis and peripheral tolerance in the immune system, and is mediated by Fas ligand (FasL) expression and the interaction between Fas and FasL. In the present study, we examined the role of the ubiquitin-proteasome system in AICD using T cell hybridoma N3-6-71 cells. The peptidyl aldehyde proteasome inhibitor carbobenzoxyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) blocked T cell receptor (TCR) stimulation-induced apoptosis in the T cell hybridoma. Fas and FasL gene expression and mouse FasL promoter activity following TCR stimulation were suppressed by PSI pretreatment. Deletion or point mutation of the kappaB site in the FasL promoter region did not suppress inducible FasL promoter activity effectively. PSI blocked extracellular signal-regulated kinase (ERK) activity induced by TCR stimulation, but had no effect on c-jun N-terminal kinase activation. ERK activation was essential for FasL expression and AICD. The initial tyrosine phosphorylation steps following TCR stimulation, i.e., phosphorylation of CD3zeta and Vav, were not altered by PSI. These data suggest that the ubiquitin-proteasome system has some regulatory function at an intermediate step between the initial tyrosine phosphorylation steps and ERK activation in AICD.     

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.     

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.     

Hypoxia-induced nucleophosmin protects cell death through inhibition of p53

Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1alpha. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxia-induced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.     

CD28 is not required for c-Jun N-terminal kinase activation in T cells.

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.     

Protein kinase C-theta-mediated signals enhance CD4+ T cell survival by up-regulating Bcl-xL

Productive engagement of TCR results in delivering signals required for T cell proliferation as well as T cell survival. Blocking TCR-mediated survival signals, T cells undergo apoptosis instead of proliferation upon TCR stimulation. During the activation process, T cells produce IL-2, which acts as an extrinsic survival factor. In addition, TCR stimulation results in up-regulation of Bcl-xL to enhance T cell survival intrinsically. We show in this study that protein kinase C (PKC)-theta is required for enhancing the survival of activated CD4+ T cells by up-regulating Bcl-xL. In response to TCR stimulation, CD4+ PKC-theta-/- T cells failed to up-regulate Bcl-xL, and underwent accelerated apoptosis via a caspase- and mitochondria-dependent pathway. Similar to PKC-theta-deficient primary CD4+ T cells, small interfering RNA-mediated knockdown of PKC-theta in Jurkat cells also resulted in apoptosis upon TCR stimulation. Forced expression of Bcl-xL was sufficient to inhibit apoptosis observed in PKC-theta knockdown cells. Furthermore, ectopic expression of PKC-theta stimulated a reporter gene driven by a mouse Bcl-xL promoter. Whereas an inactive form of PKC-theta or knockdown of endogenous PKC-theta led to inhibition of Bcl-xL reporter. PKC-theta-mediated activation of Bcl-xL reporter was inhibited by dominant-negative IkappaB kinase beta or dominant-negative AP-1. Thus, the PKC-theta-mediated signals may function not only in the initial activation of naive CD4+ T cells, but also in their survival during T cell activation by regulating Bcl-xL levels through NF-kappaB and AP-1 pathways.     

Perturbed homeostasis of peripheral T cells elicits decreased susceptibility to anti-CD3-induced apoptosis in prediabetic nonobese diabetic mice

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8(+) T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8(+) T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4(+)CD25(-)CD62L(+) but not CD4(+)CD25(+)CD62L(+) T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8(+) T cell AICD and increasing the number of CD4(+)CD25(+)CD62L(+) T cells in the periphery.