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T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized by adaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylation of ζ-chain–associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain–containing leukocyte protein of 76 kDa (SLP76), and extracellular signal–regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling–2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRγ-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRγ, and NFAT-GFP reporter. We found that Mincle specifically recognizes theMalasseziaspecies among 50 different fungal species tested.Malasseziais a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogatedMalasseziarecognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to α-mannose but not mannan. Thus, Mincle may recognize specific geometry of α-mannosyl residues onMalasseziaspecies and use this to distinguish them from other fungi.Malasseziaactivated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established.Malassezia-induced cytokine/chemokine production by macrophages from Mincle−/−mice was significantly impaired. In vivo inflammatory responses againstMalasseziawas also impaired in Mincle−/−mice. These results indicate that Mincle is the first specific receptor forMalasseziaspecies to be reported and plays a crucial role in immune responses to this fungus.

Gads is a Grb2-like adaptor protein expressed in hematopoietic cells. We demonstrated that mast cells from Gads(-/-) mice have selective functional defects. Bone marrow-derived mast cells from Gads(-/-) mice failed to induce Ca(2+) mobilization, degranulation and cytokine production upon cross-linking of FcepsilonRI. In vivo passive cutaneous anaphylaxis was also greatly impaired in Gads(-/-) mice. In contrast, Gads was dispensable for Toll-like receptor-mediated cytokine production in mast cells. Accordingly, mast cell-dependent resistance to acute peritoneal bacterial infection is not reduced in Gads(-/-) mice in vivo. Moreover, mature T and B cell responses and antibody production upon immunization were apparently normal in Gads(-/-) mice. Thus, inhibition of Gads in vivo would suppress the IgE-mediated allergic reaction with minimum adverse effects on both innate and acquired immune responses, and Gads could be an ideal target for the control of allergic responses.

Saito et al. describe a ring of focal adhesion molecules that surrounds T cell receptor microclusters and is essential for early T cell activation., The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro–adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro–adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro–adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro–adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

The pre-T cell receptor (TCR) is crucial for early T cell development and is proposed to function in a ligand-independent way. However, the molecular mechanism underlying the autonomous signals remains elusive. Here we show that the pre-TCR complex spontaneously formed oligomers. Specific charged residues in the extracellular domain of the pre-TCR alpha-chain mediated formation of the oligomers in vitro. Alteration of these residues eliminated the ability of the pre-TCR alpha-chain to support pre-TCR signaling in vivo. Dimerization but not raft localization of CD3epsilon was sufficient to simulate pre-TCR function and promote beta-selection. These results suggest that the pre-TCR complex can deliver its signal autonomously through oligomerization of the pre-TCR alpha-chain mediated by charged residues.

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and β-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4+T cells.

Some plasma membrane receptors in yeast are known to be internalized and degraded in lysosomes upon ligand-dependent ubiquitination. However, the role of ubiquitination in endocytosis and lysosomal degradation in higher eukaryotes has been controversial. In order to directly assess this question, we investigated the fate of chimeric molecules in which ubiquitin moiety was fused in-frame to the cytoplasmic region of membrane proteins. The chimeric proteins with the wild-type ubiquitin were endocytosed and delivered to lysosomes efficiently. Mutant ubiquitin with lysine-to-arginine substitution could still mediate endocytosis, suggesting that polyubiquitination is not required for the endocytosis. We next searched for the existence of an endocytosis signal(s) in the ubiquitin moiety, and identified a di-leucine signal, Leu43-Ile44. The Leu43-Ile44 sequence mediated endocytosis and lysosomal sorting in a Leu43-dependent manner. These results suggest that the di-leucine signal in ubiquitin can be involved in ubiquitination-mediated endocytosis and lysosomal targeting of membrane proteins.

Full activation of T cells with antigen (Ag) and antigen-presenting cells initiates effector functions and proliferation. When T cells are re-stimulated through the T cell receptor (TCR) after a primary stimulation with Ag, growth arrest and cell death are induced. Activation of a T cell clone by cross-linking of TCR induces interleukin (IL)-2 unresponsiveness and ultimately cell death. While the proliferative signal delivered by IL-2 induces c-myc, bcl-2 and cyclin D3 expression, the expression of bcl-2 and cyclin D3 is completely suppressed upon TCR stimulation. Furthermore, TCR stimulation induces a decrease in the protein levels of JAK3 and STAT5, suggesting that IL-2 unresponsiveness and growth arrest of T cells result from down-regulation of JAK3 and STAT5.

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3 zeta and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.

Macrophage-inducible C-type lectin (Mincle) is expressed mainly in macrophages and is induced after exposure to various stimuli and stresses. Here we show that Mincle selectively associated with the Fc receptor common gamma-chain and activated macrophages to produce inflammatory cytokines and chemokines. Mincle-expressing cells were activated in the presence of dead cells, and we identified SAP130, a component of small nuclear ribonucloprotein, as a Mincle ligand that is released from dead cells. To investigate whether Mincle is required for normal responses to cell death in vivo, we induced thymocyte death by irradiating mice and found that transient infiltration of neutrophils into the thymus could be blocked by injection of Mincle-specific antibody. Our results suggest that Mincle is a receptor that senses nonhomeostatic cell death and thereby induces the production of inflammatory cytokines to drive the infiltration of neutrophils into damaged tissue.

Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively.

A docking protein, Gab2, is recruited to the vicinity of the TCR complex and inhibits downstream signaling by interaction with negative regulators. However, the molecular mechanisms of this recruitment remain unclear. We have found that Gab2 associates with LAT upon TCR stimulation and that LAT is essential for Gab2 phosphorylation. By analysis of several Gab2 mutants, the c-Met binding domain (MBD) of Gab2 was found to be both necessary and sufficient for stimulation-induced LAT binding. Within the MBD domain, a novel Grb2 SH3 binding motif, PXXXR, is critical for constitutive association with Gads/Grb2. Through this association, Gab2 is recruited to the lipid raft after TCR ligation and exerts inhibitory function. The in vivo significance of this association is illustrated by the fact that T-cell responses are impaired in transgenic mice expressing wild-type Gab2 but not in mice expressing mutant Gab2 lacking the motif. Furthermore, T cells from Gab2-deficient mice showed enhanced proliferative responses upon TCR stimulation. These results indicate that Gads/Grb2-mediated LAT association is critical for the inhibitory function of Gab2, implying that Gab2 induced in stimulated T cells may exert an efficient negative feedback loop by recruiting inhibitory molecules to the lipid raft and competing with SLP-76 through Gads binding.

To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. We investigated the functional role of Gab2 in TCR-mediated signal transduction. Gab2 was phosphorylated by ZAP-70 and co-precipitated with phosphoproteins, such as ZAP-70, LAT, and CD3zeta, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas resulted in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogated the inhibitory activity of Gab2, but its inhibitory function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impaired the inhibitory activity, and the pleckstrin homology domain-deletion mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex.

SummaryWhen T cells recognize a peptide-major histocompatibility complex on antigen-presenting cells (APCs), T cell receptor microclusters (TCR-MCs) are generated and move to the center of the T cell-APC interface to form the central supramolecular activation cluster (cSMAC). cSMAC formation depends on stimulation strength and regulates T cell activation. We demonstrate that the dynein motor complex colocalized and coimmunoprecipitated with the TCR complex and that TCR-MCs moved along microtubules (MTs) toward the center of the immune synapse in a dynein-dependent manner to form cSMAC. MTs are located in close proximity to the plasma membrane at the activation site. TCR-MC velocity and cSMAC formation were impaired by dynein or MT inhibitors or by ablation of dynein expression. T cells with impaired cSMAC formation exhibited enhanced cellular activation including protein phosphorylation and interleukin-2 production. These results indicate that cSMAC formation by TCR-MC movement depends on dynein and MTs, and the movement regulates T cell activation.