Guilin Qiao

Cutting Edge: CTLA-4–B7 Interaction Suppresses Th17 Cell Differentiation

Th cells that produce IL-17 (Th17 cells) are a distinct subset of Th cells implicated in several autoimmune diseases. Although CD28–B7 interaction has been shown to be involved in Th17 differentiation in vitro, the role of CTLA-4 in controlling Th17 development is completely unknown. We report in this paper that blocking the CTLA-4–B7 interaction potentiates Th17 cell differentiation in vitro and in vivo. Furthermore, blocking CTLA-4–B7 interaction in vivo confers the susceptibility of experimental autoimmune myocarditis to CD28−/− mice or increases the severity of experimental autoimmune myocarditis in wild-type mice. The enhanced disease susceptibility is mediated by heightened Th17 responses. With these results, we are the first to demonstrate that CTLA-4–B7 interaction inhibits Th17 differentiation in vitro and in vivo and suppresses Th17-mediated autoimmunity.

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.

Focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in T cells

Heritable and acquired diseases of podocytes can result in focal and segmental glomerulosclerosis (FSGS). We modeled FSGS by passively transferring mouse podocyte-specific sheep Abs into BALB/c mice. BALB/c mice deficient in the key complement regulator, decay-accelerating factor (DAF), but not WT or CD59-deficient BALB/c mice developed histological and ultrastructural features of FSGS, marked albuminuria, periglomerular monocytic and T cell inflammation, and enhanced T cell reactivity to sheep IgG. All of these findings, which are characteristic of FSGS, were substantially reduced by depleting CD4+ T cells from Daf(-/-) mice. Furthermore, WT kidneys transplanted into Daf(-/-) recipients and kidneys of DAF-sufficient but T cell-deficient Balb/(cnu/nu) mice reconstituted with Daf(-/-) T cells developed FSGS. In contrast, DAF-deficient kidneys in WT hosts and Balb/(cnu/nu) mice reconstituted with DAF-sufficient T cells did not develop FSGS. Thus, we have described what we believe to be a novel mouse model of FSGS attributable to DAF-deficient T cell immune responses. These findings add to growing evidence that complement-derived signals shape T cell responses, since T cells that recognize sheep Abs bound to podocytes can lead to cellular injury and development of FSGS.

T Cell Activation Threshold Regulated by E3 Ubiquitin Ligase Cbl-b Determines Fate of Inducible Regulatory T Cells

E3 ubiquitin ligase Casitas–B-lineage lymphoma protein-b (Cbl-b) is critical for establishing the threshold for T cell activation and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of CD4+CD25+ inducible regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by naive CD4+CD25− T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is downregulated in vitro. Using TCR-transgenic Rag−/− mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.

E3 Ubiquitin Ligase Cbl-b Suppresses Proallergic T Cell Development and Allergic Airway Inflammation

SUMMARY E3 ubiquitin ligase Cbl-b has emerged as a gatekeeper that controls the activation threshold of the T cell antigen receptor and maintains the balance between tolerance and autoimmunity. Here, we report that the loss of Cbl-b facilitates T helper 2 (Th2) and Th9 cell differentiation in vitro. In a mouse model of asthma, the absence of Cbl-b results in severe airway inflammation and stronger Th2 and Th9 responses. Mechanistically, Cbl-b selectively associates with Stat6 upon IL-4 ligation and targets Stat6 for ubiquitination and degradation. These processes are heightened in the presence of T cell receptor (TCR)/ CD28 costimulation. Furthermore, we identify K108 and K398 as Stat6 ubiquitination sites. Intriguingly, introducing Stat6 deficiency into Cblb−/− mice abrogates hyper-Th2 responses but only partially attenuates Th9 responses. Therefore, our data reveal a function for Cbl-b in the regulation of Th2 and Th9 cell differentiation.

Negative Regulation of CD40-Mediated B Cell Responses by E3 Ubiquitin Ligase Casitas-B-Lineage Lymphoma Protein-B

It has been documented that CD40 is essential for B cell function. Casitas-B-lineage lymphoma protein-b (Cbl-b), an adapter protein and ubiquitin ligase, has been shown to regulate the activation of T and B cells through their Ag receptors. In this study, we report that CD40-induced B cell proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b−/− mice display enhanced thymus-dependent Ab responses and germinal center formation, whereas introduction of CD40 deficiency abolishes these effects. Hyper thymus-dependent humoral response in Cbl-b−/− mice is in part due to an intrinsic defect in B cells. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-κB and JNK. Cbl-b associates with TNF receptor-associated factor 2 upon CD40 ligation, and inhibits the recruitment of TNF receptor-associated factor 2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-κB and JNK activation, thereby suppressing B cell responses.

TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser(473) in the hydrophobic motif, along with Thr(308) in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr(308), but the kinase(s) responsible for phosphorylating Akt at Ser(473) (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser(473) phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser(473) in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

E3 Ubiquitin Ligase Cbl-b Regulates Thymic-Derived CD4+CD25+ Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination

CD28 costimulation is essential for the development of thymic-derived CD4+CD25+Foxp3+ regulatory T cells (“tTregs”). E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. In this paper, we report that the loss of Cbl-b partially but significantly rescues the defective development of tTregs in Cd28−/− mice. This partial rescue is independent of IL-2. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in Cd28−/− T cells, the defective development of tTregs in Cd28−/− mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Stub1 and Cbl-b. Treating Cd28−/− mice with a proteasome inhibitor completely rescues defective tTreg development in these mice. Therefore, Cbl-b, together with Stub1, ubiquitinate Foxp3, and regulate tTreg development.

Altered thymic selection by overexpressing cellular FLICE inhibitory protein in T cells causes lupus-like syndrome in a BALB/c but not C57BL/6 strain

The cellular FLICE inhibitory protein (c-FLIP) is an endogenous inhibitor of the caspase-8 proapoptotic signaling pathway downstream of death receptors. Recent evidence indicates that the long form of c-FLIP (c-FLIPL) is required for proliferation and effector T-cell development. However, the role of c-FLIPL in triggering autoimmunity has not been carefully analyzed. We now report that c-FLIPL transgenic (Tg) mice develop splenomegaly, lymphadenopathy, multiorgan infiltration, high titers of auto-antibodies, and proliferative glomerulonephritis with immune complex deposition in a strain-dependent manner. The development of autoimmunity requires CD4+ T cells and may result from impaired thymic selection. At the molecular level, c-FLIPL overexpression inhibits the ζ chain-associated protein tyrosine kinase of 70 kDa (ZAP-70) activation, thus impairing the signaling pathway derived from ZAP-70 required for thymic selection. Therefore, we have identified c-FLIPL as a susceptibility factor under the influence of epistatic modifiers for the development of autoimmunity.

Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism.

Program death-1 (PD-1) has been documented to negatively regulate immune responses. However, the cellular and molecular mechanisms for PD-1-mediated immune suppression have not been fully elucidated. In this study, we show that loss of PD-1 does not lead to defective induction of CD4(+) T cell anergy in vitro and in vivo. Rather, the absence of PD-1 inhibits the development of inducible CD4(+)Foxp3(+) regulatory T cells (iTregs) induced by TGF-β in vitro. In support of this finding, PD-1 deficiency impairs the generation of iTregs in vivo and leads to development of severe T cell-transfer-induced colitis. Mechanistically, defective iTreg generation in the absence of PD-1 was attributed to the heightened phosphorylation of Akt. Therefore, we first demonstrate that PD-1 controls peripheral T cell tolerance via an anergy-independent but iTreg-dependent mechanism.