Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.

Giant cell arteritis (GCA) is a granulomatous and occlusive vasculitis that causes blindness, stroke, and aortic aneurysm. CD4+ T cells are selectively activated in the adventitia of affected arteries. In human GCA artery–severe combined immunodeficiency (SCID) mouse chimeras, depletion of CD83+ dendritic cells (DCs) abrogated vasculitis, suggesting that DCs are critical antigen-presenting cells in GCA. Healthy medium-size arteries possessed an indigenous population of DCs at the adventitia–media border. Adoptive T cell transfer into temporal artery–SCID mouse chimeras demonstrated that DCs in healthy arteries were functionally immature, but gained T cell stimulatory capacity after injection of lipopolysaccharide. In patients with polymyalgia rheumatica (PMR), a subclinical variant of GCA, adventitial DCs were mature and produced the chemokines CCL19 and CCL21, but vasculitic infiltrates were lacking. Human histocompatibility leukocyte antigen class II–matched healthy arteries, PMR arteries, and GCA arteries were coimplanted into SCID mice. Immature DCs in healthy arteries failed to stimulate T cells, but DCs in PMR arteries could attract, retain, and activate T cells that originated from the GCA lesions. We propose that in situ maturation of DCs in the adventitia is an early event in the pathogenesis of GCA. Activation of adventitial DCs initiates and maintains T cell responses in the artery and breaks tissue tolerance in the perivascular space.

Immature dendritic cells (DCs) are scattered throughout peripheral tissues and act as sentinels that sample the antigenic environment. After activation, they modify their chemokine receptor profile and migrate toward lymphoid tissues. On arrival, they have matured into chemokine-producing DCs that express co-stimulatory molecules and can prime naive T cells. Normal temporal arteries contain immature DCs that are located at the media-adventitia border. In temporal arteries affected by giant cell arteritis, DCs are highly enriched and activated and have matured into fully differentiated cells producing the chemokines, CCL18, CCL19, and CCL21. In keeping with their advanced maturation, DCs in the granulomatous lesions possess the chemokine receptor, CCR7. CCR7 binds CCL19 and CCL21, causing the highly activated DCs to be trapped in the peripheral tissue site. The co-stimulatory molecule, CD86, which is critical for DC/T-cell interaction, is expressed by a subset of DCs captured in the arterial wall. DC/T-cell interaction does not involve interleukin-12; transcripts for interleukin-12 p40 are absent in the vasculitic infiltrates. We propose that differentiation of DCs and the autocrine and paracrine actions of chemokines in granulomatous lesions misdirect DCs away from their usual journey to lymphoid organs and are critical in maintaining T-cell activation and granuloma formation in giant cell arteritis.

Engagement of the T cell antigen receptor (TCR)1 by the antigenic peptide plus major histocompatibility complex (MHC) in the antigen presenting cells triggers rapid tyrosine phosphorylation and activation of protein-tyrosine kinases, the Src family (Lck and Fyn), and the Syk family (Zap-70). Activation of these kinases in turn induces tyrosine phosphorylation of a number of intracellular substrates including adaptor proteins to form intermolecular network (1, 2). Cbl is one of the adaptor proteins, which consists of an amino-terminal SH2-like domain, a RING finger, and carboxyl-terminal proline-rich sequences with potential tyrosine phosphorylation sites (3). Previous studies from numerous laboratories have demonstrated that Cbl associates with a number of critical signaling molecules upon T cell activation including Zap-70, Grb-2, 14-3-3, phosphatidylinositol 3-kinase (PI3K), and Crk-L (3). In addition to its adaptor's role, Cbl also functions as an E3 ubiquitin (Ub) ligase, whose RING finger domain binds to a Ub-loaded conjugation enzyme or E2, and whose other protein interaction domains recruit potential substrates; Cbl then helps transfer Ub from the E2 to the substrate (4, 5). The identification of Cbl as an E3 has facilitated our understanding of Cbl in the regulation of intracellular signaling transduction.Cbl-b was isolated as a mammalian Cbl homologue from human breast caner cells and it is ubiquitously expressed and has a very high homology with Cbl, particularly in the amino terminus and the RING finger domain (6). The importance of Cbl-b in T cell regulation is underscored by the increased proliferation and cytokine production in Cbl-b-deficient T cells (7,8). Cbl-b Ϫ/Ϫ mice also display spontaneous or antigen-induced autoimmunity. We previously showed that Cbl-b has E3 ligase activity by inducing ubiquitionation of p85, the regulatory subunit of PI3K (9). Instead of inducing degradation of p85, Ub conjugation to p85 affects its interaction with upstream molecules such as TCR chain and CD28, thus regulating the protein complex formation (10). In human Jukat T cell line, we observed that Cbl-b becomes phosphorylated on tyrosine residues and associates with Crk-L in an activationdependent manner (11). However, the physiological significance of Cbl-b and Crk-L interaction remains unclear.Crk-L is an adaptor protein that is composed of an NH 2 -terminal SH2 domain and COOH-terminal two SH3 domains. The SH2 domain has been shown to bind to Cbl and/or Cbl-b in an activation-dependent manner, whereas a COOH-terminal SH3 domain constitutively associates with C3G, a guanine exchange factor, which has been shown to be a specific exchange factor for Rap-1 (12). The Cbl-Crk-L-C3G signal pathway has been proposed to play a role in Rap-1 activation and therefore in negative regulation of Ras signal pathway in T cells (13). Whether Cbl-b is also involved in Rap1 activation in T cells remains to be determined. In this study, we examined whether Cbl-b acts as an E3 ligase for Crk-L and whether Cbl-b deficiency affect...

Generation of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) induces the proliferation and migration of adipose-derived stem cells (ASCs). However, the functional role of mitochondrial ROS (mtROS) generation in ASCs is unknown. Therefore, we have investigated whether hypoxia induces the differentiation of ASCs via ROS generation. We also have tried to identify the cellular mechanisms of ROS generation underlying adipocyte differentiation. Hypoxia (2%) and ROS generators, such as antimycin and rotenone, induced adipocyte differentiation, which was attenuated by an ROS scavenger. Although Nox4 generates ROS and regulates proliferation of ASCs, Nox4 inhibition or Nox4 silencing did not inhibit adipocyte differentiation; indeed fluorescence intensity of mito-SOX increased in hypoxia, and treatment with mito-CP, a mtROS scavenger, significantly reduced hypoxia-induced adipocyte differentiation. Phosphorylation of Akt and mTOR was induced by hypoxia, while inhibition of these molecules prevented adipocyte differentiation. Thus hypoxia induces adipocyte differentiation by mtROS generation, and the PI3K/Akt/mTOR pathway is involved.