Zuoming Sun

Stabilized β-Catenin Extends Thymocyte Survival by Up-Regulating Bcl-xL

CD4+CD8+ double-positive (DP) thymocytes, which are extremely sensitive to apoptosis, specifically up-regulate Bcl-xL to extend their lifespan. Deletion of the Bcl-xL gene leads to premature apoptosis of the thymocytes. In this study, we show that stabilization of β-catenin, a critical coactivator for T cell factor (TCF), enhances DP thymocyte survival via up-regulating Bcl-xL. Spontaneous or glucocorticoid-induced thymocyte apoptosis was associated with reduced levels of β-catenin and Bcl-xL. Transgenic expression of a stabilized β-catenin protected DP thymocytes from both spontaneous and glucocorticoid-induced apoptosis, resulting in significantly increased thymic cellularity. Compared with the wild-type mice, both protein and transcript levels of Bcl-xL were significantly increased in thymocytes of β-catenin transgenic mice. In addition, TCF-1 as well as β-catenin were able to stimulate transcriptional activity of the reporter driven by a Bcl-xL promoter. β-Catenin/TCF is thus able to act as a signal to up-regulate Bcl-xL levels in DP thymocytes, resulting in their enhanced survival.

Protein Kinase C-θ-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)-θ is required for enhancing the survival of activated CD4+ T cells by up-regulating Bcl-xL. In response to TCR stimulation, CD4+ PKC-θ−/− T cells failed to up-regulate Bcl-xL, and underwent accelerated apoptosis via a caspase- and mitochondria-dependent pathway. Similar to PKC-θ-deficient primary CD4+ T cells, small interfering RNA-mediated knockdown of PKC-θ in Jurkat cells also resulted in apoptosis upon TCR stimulation. Forced expression of Bcl-xL was sufficient to inhibit apoptosis observed in PKC-θ knockdown cells. Furthermore, ectopic expression of PKC-θ stimulated a reporter gene driven by a mouse Bcl-xL promoter. Whereas an inactive form of PKC-θ or knockdown of endogenous PKC-θ led to inhibition of Bcl-xL reporter. PKC-θ-mediated activation of Bcl-xL reporter was inhibited by dominant-negative IκB kinase β or dominant-negative AP-1. Thus, the PKC-θ-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-κB and AP-1 pathways.

T-cell receptor-induced NF-κB activation is negatively regulated by E3 ubiquitin ligase Cbl-b

ABSTRACT It has previously been shown that E3 ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) negatively regulates T-cell activation, but the molecular mechanism(s) underlying this inhibition is not completely defined. In this study, we report that the loss of Cbl-b selectively results in aberrant activation of NF-κB upon T-cell antigen receptor (TCR) ligation, which is mediated by phosphatidylinositol 3-kinase (PI3-K)/Akt and protein kinase C-θ (PKC-θ). TCR-induced hyperactivation of Akt in the absence of Cbl-b may potentiate the formation of caspase recruitment domain-containing membrane-associated guanylate kinase protein 1 (CARMA1)-B-cell lymphoma/leukemia 10 (Bcl10)-mucosa-associated lymphatic tissue 1(MALT1) (CBM) complex, which appears to be independent of PKC-θ. Cbl-b associates with PKC-θ upon TCR stimulation and regulates TCR-induced PKC-θ activation via Vav-1, which couples PKC-θ to PI3-K and allows it to be phosphorylated. PKC-θ then couples IκB kinases (IKKs) to the CBM complex, resulting in the activation of the IKK complex. Therefore, our data provide the first evidence to demonstrate that the down-regulation of TCR-induced NF-κB activation by Cbl-b is mediated coordinately by both Akt-dependent and PKC-θ-dependent signaling pathways in primary T cells.

RORγt Recruits Steroid Receptor Coactivators to Ensure Thymocyte Survival

Thymocytes undergo apoptosis unless a functional TCR is assembled. Steroid receptor coactivators (SRCs) regulate nuclear receptor-mediated transcription by associated histone acetyltransferase activity. However, it has been a challenge to demonstrate the in vivo function of SRCs due to the overlapping functions among different members of SRCs. In this study, we show that recruitment of SRCs is required for thymic-specific retinoic acid-related orphan receptor γ (RORγ)t-regulated thymocyte survival in vivo. An activation function 2 domain, identified at the carboxyl terminus of RORγt, is responsible for recruiting SRCs. A mutation in the activation function domain (Y479F) of RORγt disrupted the interaction with SRCs and abolished RORγt-mediated trans-activation but not its ability to inhibit transcription. Transgenes encoding the wild-type RORγt, but not the mutant, restored thymocyte survival in RORγ null mice. Our results thus clearly demonstrate that RORγt recruits SRCs to impose a gene expression pattern required to expand the life span of thymocytes in vivo, which increases the opportunities for assembling a functional TCR.

Transcriptional Regulation of CD4 Gene Expression by T Cell Factor-1/β-Catenin Pathway

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/β-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/β-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1−/− DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1−/− cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with β-catenin. Furthermore, forced expression of a stabilized β-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized β-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and β-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/β-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

The critical role of protein kinase C-θ in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-θ (PKC-θ), superantigen (staphylococcal enterotoxin B)-induced deletion of Vβ8+ CD4+ T cells was defective in PKC-θ−/− mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-θ−/− mice. PKC-θ is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-θ-mediated activation of NF-AT pathway. In addition, PKC-θ−/− T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-θ, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-θ−/− T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-θ is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.

A Critical Role for Protein Kinase C-θ-Mediated T Cell Survival in Cardiac Allograft Rejection

Protein kinase C (PKC)-θ mediates the critical TCR signals required for T cell activation. Previously, we have shown that in response to TCR stimulation, PKC-θ−/− T cells undergo apoptosis due to greatly reduced levels of the anti-apoptotic molecule, Bcl-xL. In this study, we demonstrate that PKC-θ-regulated expression of Bcl-xL is essential for T cell-mediated cardiac allograft rejection. Rag1−/− mice reconstituted with wild-type T cells readily rejected fully mismatched cardiac allografts, whereas Rag1−/− mice reconstituted with PKC-θ−/− T cells failed to promote rejection. Transgenic expression of Bcl-xL in PKC-θ−/− T cells was sufficient to restore cardiac allograft rejection, suggesting that PKC-θ-regulated survival is required for T cell-mediated cardiac allograft rejection in this adoptive transfer model. In contrast to adoptive transfer experiments, intact PKC-θ−/− mice displayed delayed, but successful cardiac allograft rejection, suggesting the potential compensation for PKC-θ function. Finally, a subtherapeutic dose of anti-CD154 Ab or CTLA4-Ig, which was not sufficient to prevent cardiac allograft rejection in the wild-type mice, prevented heart rejection in the PKC-θ−/− mice. Thus, in combination with other treatments, inhibition of PKC-θ may facilitate achieving long-term survival of allografts.

Mechanisms regulating the development and function of natural regulatory T cells

The key of the immune system is to protect the host from foreign threat posed by pathogens and from the internal threat posed by self-attacking lymphocytes. The ability to discriminate self versus non-self ensures that only “non-self” pathogens, but not the self antigens, are attacked. Such tolerance to “self” arises from the central tolerance mechanisms that include the deletion of thymocytes with high reactivity to self antigens and also the induction of unresponsiveness of autoreactive T cells in the periphery. Natural regulatory T cells (nTregs) directly inhibit effector T cells, and keep their proliferation in control. Apart from preventing autoimmune reactions, Tregs also contribute to peripheral immune homeostasis as evidenced by the excessive lymphocyte accumulation in peripheral lymphoid organs and intestinal inflammation in the absence of nTregs. Here we discuss the molecular aspects of the development and suppressive function of naturally occurring Tregs. Accumulating evidence shows the importance of these Tregs in autoimmunity, tumor immunity, organ transplantation, allergy, and microbial immunity.

Requirement of Calcineurin Aβ for the Survival of Naive T Cells

Calcineurin (Cn) is a Ca2+/calmodulin-dependent phosphatase that dephosphorylates and activates NFAT, a transcription factor essential for T cell activation. T lymphocytes predominantly express the calcineurin Aβ (CnAβ) isoform, and the deletion of the CnAβ gene results in defective T cell proliferation and IL-2 production in response to TCR stimulation. In this study, we show that CnAβ enhances the spontaneous survival of naive T cells by maintaining high levels of Bcl-2, a critical homeostatic survival factor for naive T cells. T cells obtained from CnAβ−/− mice displayed accelerated spontaneous apoptosis. The observed apoptosis of the CnAβ−/− T cells was prevented by IL-7 and IL-15, two cytokines critical for the homeostatic survival of naive T cells. Furthermore, CD4+ or CD8+ single positive CnAβ−/− thymocytes also underwent accelerated apoptosis. However, no obvious difference in the apoptosis of CD4+CD8+ double positive thymocytes was observed between CnAβ−/− and wild-type mice, suggesting a specific function of CnAβ in the survival of single positive T cells. Bcl-2 levels were found to be significantly lower in CnAβ−/− T cells. Transgenic expression of Bcl-xL restored the survival of the CnAβ−/− T cells. Thus, in addition to its role in mediating TCR signals essential for T cell activation, CnAβ is also required for the homeostatic survival of naive T cells.