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The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a multimeric, cytoplasmic, protein complex that regulates maturation and secretion of interleukin (IL)-1β, a potent pro-inflammatory cytokine. Critical to host defense against pathogens, IL-1β amplifies early innate immune responses by activating transcription of numerous other cytokines and chemokines. Excessive IL-1β is associated with poor outcomes in inflammatory illnesses, such as sepsis and the acute respiratory distress syndrome (ARDS). Tight regulation of this signaling axis is vital, but little is known about mechanisms to limit excessive inflammasome activity. Here we identify the deubiquitinase STAM-binding protein (STAMBP) as a negative regulator of the NLRP3 inflammasome. In monocytes, knockout of STAMBP by CRISPR/Cas9 gene editing increased expression of numerous cytokines and chemokines in response to Toll-like receptor (TLR) agonists or bacterial lipopolysaccharide (LPS). This exaggerated inflammatory response was dependent on IL-1β signaling, and STAMBP knockout directly increased release of IL-1β with TLR ligation. While STAMBP does not modulate NLRP3 protein abundance, cellular depletion of the deubiquitinase increased NLRP3 K63 chain polyubiquitination resulting in increased NLRP3 inflammasome activation. These findings describe a unique mechanism of non-degradative ubiquitination of NLRP3 by STAMBP to limit excessive inflammasome activation and to reduce injurious IL-1β signaling., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Trafficking of toll-like receptor 3 (TLR3) from the endoplasmic reticulum (ER) to endolysosomes and its subsequent proteolytic cleavage are required for it to sense viral double-stranded RNA (dsRNA) and trigger antiviral response, yet the underlying mechanisms remain enigmatic. We show that the E3 ubiquitin ligase TRIM3 is mainly located in the Golgi apparatus and transported to the early endosomes upon stimulation with the dsRNA analog poly(I:C). TRIM3 mediates K63-linked polyubiquitination of TLR3 at K831, which is enhanced following poly(I:C) stimulation. The polyubiquitinated TLR3 is recognized and sorted by the ESCRT (endosomal sorting complex required for transport) complexes to endolysosomes. Deficiency of TRIM3 impairs TLR3 trafficking from the Golgi apparatus to endosomes and its subsequent activation. Trim3 -/- cells and mice express lower levels of antiviral genes and show lower levels of inflammatory response following poly(I:C) but not lipopolysaccharide (LPS) stimulation. These findings suggest that TRIM3-mediated polyubiquitination of TLR3 represents a feedback-positive regulatory mechanism for TLR3-mediated innate immune and inflammatory responses., Competing Interests: The authors declare no competing interest.

In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV., (Copyright © 2019. Published by Elsevier Ltd.)

Proximity ligation assay (PLA) is a newly developed technique that outperforms the traditional immunoassays for visualizing the in situ endogenous protein-protein interactions and localizations and the activation of proteins in cell culture systems as well as in tissue sections. PLA, when combined with cellular marker staining, becomes a powerful approach to identify differential interaction of the proteins of endosomal sorting complex required for transport (ESCRT) at distinct stages of virus infection. In this chapter, we describe a PLA protocol to study the localization and interaction between the ESCRT protein TSG101 and endosomal markers in early stages of viral endocytosis in in vitro infected cells.

Many enveloped viruses utilize the cellular ESCRT pathway for budding, even flaviviruses, which form viral particles inside replication organelles derived from the endoplasmic reticulum (ER). In this section, we introduce methods for detecting several ESCRT subunit proteins in virus-infected cells by immunofluorescence microscopy and immunoelectron microscopy (immuno-EM). We also introduce a new method; correlative light microscopy and electron microscopy (CLEM), which allows the observation of target structures with both high-resolution EM and fluorescence labeling.

Most endosomal sorting complex required for transport (ESCRT)-III proteins are not fully functional when expressed as fusion of fluorescent or epitope tags, frequently making the use of specific antibodies the only available method for their detection. Heterologous expression of ESCRT-III proteins in bacteria often results in the formation of insoluble aggregates or inclusion bodies that interfere with their purification. However, inclusion bodies are usually pure protein aggregates with high antigenicity. In addition, since proteins within inclusion bodies are presented in a range of folding states, immunization with inclusion bodies can potentially result in antibodies with specificity for different folding states of the protein under study. We describe here a protocol to isolate bacterial inclusion bodies of plant ESCRT-III proteins for production of polyclonal antibodies. We also describe a nitrocellulose-based immunoaffinity purification method that allows the immobilization of ESCRT-III proteins and the subsequent isolation of specific antibodies from a crude serum.

Anaphylactic reactions are triggered when allergens enter the blood circulation and activate immunoglobulin E (IgE)-sensitized mast cells (MCs), causing systemic discharge of prestored proinflammatory mediators. As MCs are extravascular, how they perceive circulating allergens remains a conundrum. Here, we describe the existence of a CD301b + perivascular dendritic cell (DC) subset that continuously samples blood and relays antigens to neighboring MCs, which vigorously degranulate and trigger anaphylaxis. DC antigen transfer involves the active discharge of surface-associated antigens on 0.5- to 1.0-micrometer microvesicles (MVs) generated by vacuolar protein sorting 4 (VPS4). Antigen sharing by DCs is not limited to MCs, as neighboring DCs also acquire antigen-bearing MVs. This capacity of DCs to distribute antigen-bearing MVs to various immune cells in the perivascular space potentiates inflammatory and immune responses to blood-borne antigens., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non-structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11-linked poly-ubiquitin chains from caspase-1 at Lys134, thus inhibiting the proteasomal degradation of caspase-1. The enhanced stabilization of caspase-1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKV in vitro and in vivo Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future., (© 2018 The Authors.)

The activation of interferon (IFN)-regulatory factor-3 (IRF3), characterized by phosphorylation and nuclear translocation of the latent transcription factor, is central to initiating innate antiviral responses. Whereas much has been learned about the upstream pathways and signaling mechanisms leading to IRF3 activation, how activated IRF3 operates in the nucleus to control transcription of IFNs remains obscure. Here we identify EAP30 (a.k.a, SNF8/VPS22), an endosomal sorting complex required for transport (ESCRT)-II subunit, as an essential factor controlling IRF3-dependent antiviral defense. Depletion of EAP30, but not other ESCRT-II subunits, compromised IRF3-dependent induction of type I and III IFNs, IFN-stimulated genes (ISGs) and chemokines by double-stranded RNA or viruses. EAP30, however, was dispensable for the induction of inflammatory mediators of strict NF-κB target. Significantly, knockdown of EAP30 also impaired the establishment of an antiviral state against vesicular stomatitis virus and hepatitis C virus, which are of distinct viral families. Mechanistically, EAP30 was not required for IRF3 activation but rather acted at a downstream step. Specifically, a fraction of EAP30 localized within the nucleus, where it formed a complex with IRF3 and its transcriptional co-activator, CREB-binding protein (CBP), in a virus-inducible manner. These interactions promoted IRF3 binding to target gene promoters such as IFN-β, IFN-λ1 and ISG56. Together, our data describe an unappreciated role for EAP30 in IRF3-dependent innate antiviral response in the nucleus.

Enterovirus 71 (EV71) is an RNA virus that causes hand-foot-mouth disease (HFMD), and even fatal encephalitis in children. Although EV71 pathogenesis remains largely obscure, host immune responses may play important roles in the development of diseases. Recognition of pathogens mediated by Toll-like receptors (TLRs) induces host immune and inflammatory responses. Intracellular TLRs must traffic from the endoplasmic reticulum (ER) to the endolysosomal network from where they initiate complete signaling, leading to inflammatory response. This study reveals a novel mechanism underlying the regulation of TLR7 signaling during EV71 infection. Initially, we show that multiple cytokines are differentially expressed during viral infection and demonstrate that EV71 infection induces the production of proinflammatory cytokines through regulating TLR7-mediated p38 MAPK, and NF-κB signaling pathways. Further studies reveal that the expression of the endosome-associated protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is upregulated and highly correlated with the expression of TLR7 in EV71 infected patients, mice, and cultured cells. Virus-induced HRS subsequently enhances TLR7 complex formation in early- and late-endosome by interacting with TLR7 and TAB1. Moreover, HRS is involved in the regulation of the TLR7/NF-κB/p38 MAPK and the TLR7/NF-κB/IRF3 signaling pathways to induce proinflammatory cytokines and interferons, respectively, resulting in the orchestration of inflammatory and immune responses to the EV71 infection. Therefore, this study demonstrates that HRS acts as a key component of TLR7 signaling to orchestrate immune and inflammatory responses during EV71 infection, and provides new insights into the mechanisms underlying the regulation of host inflammation and innate immunity during EV71 infection.

The acquisition of a protective vertebrate immune system hinges on the efficient generation of a diverse but self-tolerant repertoire of T cells by the thymus through mechanisms that remain incompletely resolved. Here we identified the endosomal-sorting-complex-required-for-transport (ESCRT) protein CHMP5, known to be required for the formation of multivesicular bodies, as a key sensor of thresholds for signaling via the T cell antigen receptor (TCR) that was essential for T cell development. CHMP5 enabled positive selection by promoting post-selection thymocyte survival in part through stabilization of the pro-survival protein Bcl-2. Accordingly, loss of CHMP5 in thymocyte precursor cells abolished T cell development, a phenotype that was 'rescued' by genetic deletion of the pro-apoptotic protein Bim or transgenic expression of Bcl-2. Mechanistically, positive selection resulted in the stabilization of CHMP5 by inducing its interaction with the deubiquitinase USP8. Our results thus identify CHMP5 as an essential component of the post-translational machinery required for T cell development.

Inflammasomes regulate innate immune responses by facilitating maturation of inflammatory cytokines, interleukin (IL)-1β and IL-18. NACHT, LRR and PYD domains-containing protein 7 (NALP7) is one inflammasome constituent, but little is known about its cellular handling. Here we show a mechanism for NALP7 protein stabilization and activation of the inflammasome by Toll-like receptor (TLR) agonism with bacterial lipopolysaccharide (LPS) and the synthetic acylated lipopeptide Pam3CSK4. NALP7 is constitutively ubiquitinated and recruited to the endolysosome for degradation. With TLR ligation, the deubiquitinase enzyme, STAM-binding protein (STAMBP) impedes NALP7 trafficking to lysosomes to increase NALP7 abundance. STAMBP deubiquitinates NALP7 and STAMBP knockdown abrogates LPS or Pam3CSK4-induced increases in NALP7 protein. A small-molecule inhibitor of STAMBP deubiquitinase activity, BC-1471, decreases NALP7 protein levels and suppresses IL-1β release after TLR agonism. These findings describe a unique pathway of inflammasome regulation with the identification of STAMBP as a potential therapeutic target to reduce pro-inflammatory stress.

HIV infection presents a major community health hazard, partially because the HIV virus is capable of evading antiretroviral therapies. Most anti-HIV drugs were intended to target virus-encoded mechanisms; however, some host-encoded molecules comparatively execute a vital role in the life cycle of virus. Thus, these might be considered as target sites for antiviral agents. TSG101 is important among these antiviral therapies because, as a cytoplasmic molecule, it facilitates viral budding and release. In this review, HIV-infected cells have TSG101 on their surface and thus can be used in antibody-based therapies. The development of a monoclonal antibody CB8-2 lessens the assembly of viruses from infected cells. This mechanism represents the potential use of TSG101-directed antibodies to fight against AIDS.

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3β. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.

Epithelial barrier dysfunction is associated with a number of inflammatory disorders. However, the pathogenesis of epithelial barrier dysfunction is unclear. This study aims to elucidate the involvement of dicaine in airway epithelial barrier dysfunction. In the present study, an RPMI2650 (Rpc) human airway epithelial cell line was cultured, with or without dicaine, in monolayers using Transwells. In order to assess airway epithelial barrier function, the levels of transepithelial electrical resistance and permeability to ovalbumin (OVA) were measured. Expression of apoptosis-linked gene 2-interacting protein X (Alix) in Rpc cells was assessed using quantitative reverse transcription‑polymerase chain reaction and western blotting. The antigenicity of OVA was assessed using a T cell proliferation assay. The results of the present study demonstrated that Alix expression levels were markedly lower in Rpc cells treated with dicaine, compared with those not treated with dicaine. An increase in the level of transcellular permeability to OVA was observed in Rpc monolayers following treatment with dicaine, compared with that in the Rpc monolayer without dicaine treatment. Furthermore, the Rpc monolayer maintained high antigenicity and induced antigen specific T cell proliferation. In conclusion, dicaine causes a decrease in expression levels of Alix, which resulted in compromise of the Rpc cell monolayer epithelial barrier function.

For the initiation of adaptive immune responses, dendritic cells present antigenic peptides in association with major histocompatibility complex class II (MHCII) to naïve CD4(+) T lymphocytes. In this review, we discuss how antigen presentation is regulated through intracellular processing and trafficking of MHCII. Newly synthesized MHCII is chaperoned by the invariant chain to endosomes, where peptides from endocytosed pathogens can bind. In nonactivated dendritic cells, peptide-loaded MHCII is ubiquitinated and consequently sorted by the ESCRT machinery to intraluminal vesicles of multivesicular bodies, ultimately leading to lysosomal degradation. Ubiquitination of newly synthesized MHCII is blocked when dendritic cells are activated, now allowing its transfer to the cell surface. This mode of regulation for MHCII is a prime example of how molecular processing and sorting at multivesicular bodies can determine the expression of signaling receptors at the plasma membrane.

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

Aim: Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus., Methods: The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot., Results: We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated., Conclusion: CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

The bacteria inhabiting the mammalian gastrointestinal (GI) tract play a vital role in normal digestion and immune function. In a healthy host, the immune system is tolerant to gut bacteria and does not mount an effector response to bacteria-derived antigens. Loss of tolerance to intestinal microflora has been associated with inflammatory bowel disease (IBD) in both mice and humans. Mice lacking Ndfip1, an adaptor protein for E3 ubiquitin ligases of the Nedd4-family, in T cells (Ndfip1-cKO) develop a disease resembling IBD. Inflammation in these mice is characterized by increased activation of peripheral T cells, infiltration of eosinophils into the GI tract, and epithelial hypertrophy in the esophagus. We hypothesized that this intestinal inflammation in Ndfip1-cKO mice is caused by a loss of T-cell tolerance to bacterial antigens. Here, we show that treatment of Ndfip1-cKO mice with broad-spectrum antibiotics drastically reduced bacterial load in stool but had little effect on T-cell activation and did not affect eosinophil infiltration into the GI tract or epithelial hypertrophy in the esophagus. Thus, inflammation in Ndfip1-cKO mice is not caused by a loss of tolerance to intestinal microbiota. Rather, T cell activation and eosinophilia may instead be triggered by other environmental antigens.

The molecular mechanisms regulating noncanonical protein transport across cellular membranes are poorly understood. Cross-presentation of exogenous antigens on MHC I molecules by dendritic cells (DCs) generally requires antigen translocation from the endosomal compartment into the cytosol for proteasomal degradation. In this study, we demonstrate that such translocation is controlled by the endocytic receptor and regulated by ubiquitination. Antigens internalized by the mannose receptor (MR), an endocytic receptor that targets its ligands specifically toward cross-presentation, were translocated into the cytosol only after attachment of a lysin48-linked polyubiquitin chain to the cytosolic region of the MR. Furthermore, we identify TSG101 as a central regulator of MR ubiquitination and antigen translocation. Importantly, we demonstrate that MR polyubiquitination mediates the recruitment of p97, a member of the ER-associated degradation machinery that provides the driving force for antigen translocation, toward the endosomal membrane, proving the central role of the endocytic receptor and its ubiquitination in antigen translocation.

Endosomal sorting complex required for transport (ESCRT)-III subunit charged multivesicular body protein 2B (CHMP2B) is involved in the degradation of proteins in the endocytic and autophagic pathways. Mutations in the CHMP2B gene are reportedly associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) characterised by accumulation of ubiquitinated protein aggregates in affected neurons, suggesting a relationship between protein accumulation and efficient autophagic degradation. This study investigated CHMP2B immunoreactivity in the hippocampus of patients with Alzheimer's disease (AD), revealing intense labeling of intraneuronal dot-like structures by antibody to CHMP2B. Since the morphological characteristics of these granular structures were compatible with those of granulovacuolar degeneration (GVD), a hallmark of AD pathology, immunohistochemical study using anti-CHMP2B antibody was performed using AD and control brain sections to investigate whether this antibody can be used as a GVD label. The number and percentage of hippocampal neurons with CHMP2B-positive granules were higher in AD cases and CHMP2B-positive granules corresponded to GVD. Anti-CHMP2B antibody detected a single 28-kDa band on Western blotting using control and AD specimens. This antibody clearly and intensely detected GVD over the hippocampus and entorhinal and transentorhinal cortices. These findings suggest that researchers will be able to use CHMP2B as a molecular label for studying GVD., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.

Agonist MHC-peptide complexes in the immunological synapse (IS) signal through T cell receptor (TCR) microclusters (MCs) that converge into a central supramolecular activation cluster (cSMAC). The determinants and function of the cSMAC remain unknown. We demonstrate an essential role for ubiquitin (Ub) and TSG101, but less so for HRS, in signal processing events at the cSMAC. Using siRNA in primary T cells, we show that Ub recognition by TSG101 is required for cSMAC formation, TCR MC signal termination, TCR downregulation, and segregation of TCR-MHC-peptide from PKC-theta-enriched signaling complexes. Weak agonist MHC-peptide induced CD80-dependent TCR MCs that dissociated in the center of the IS without recruiting TSG101. These results support TSG101-dependent recognition of CD80-independent TCR MCs as a molecular checkpoint for TCR downregulation., (Copyright 2010 Elsevier Inc. All rights reserved.)