The K48-K63 Branched Ubiquitin Chain Regulates NF-κB Signaling.

Polyubiquitin chains of different topologies regulate diverse cellular processes. K48- and K63-linked chains, the two most abundant chain types, regulate proteolytic and signaling pathways, respectively. Although recent studies reported important roles for heterogeneous chains, the functions of branched ubiquitin chains remain unclear. Here, we show that the ubiquitin chain branched at K48 and K63 regulates nuclear factor κB (NF-κB) signaling. A mass-spectrometry-based quantification strategy revealed that K48-K63 branched ubiquitin linkages are abundant in cells. In response to interleukin-1ß, the E3 ubiquitin ligase HUWE1 generates K48 branches on K63 chains formed by TRAF6, yielding K48-K63 branched chains. The K48-K63 branched linkage permits recognition by TAB2 but protects K63 linkages from CYLD-mediated deubiquitylation, thereby amplifying NF-κB signals. These results reveal a previously unappreciated cooperation between K48 and K63 linkages that generates a unique coding signal: ubiquitin chain branching differentially controls readout of the ubiquitin code by specific reader and eraser proteins to activate NF-κB signaling.

MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

Ubiquitination of MHC Class II by March-I Regulates Dendritic Cell Fitness.

The expression and turnover of Ag-specific peptide-MHC class II (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to efficiently activate CD4 T cells. Ubiquitination of pMHC-II by the E3 ubiquitin ligase March-I regulates surface expression and survival of pMHC-II in DCs. We now show that despite their high levels of surface pMHC-II, MHC class II (MHC-II) ubiquitination-deficient mouse DCs are functionally defective; they are poor stimulators of naive CD4 T cells and secrete IL-12 in response to LPS stimulation poorly. MHC-II ubiquitination-mutant DC defects are cell intrinsic, and single-cell RNA sequencing demonstrates that these DCs have an altered gene expression signature as compared with wild-type DCs. Curiously, these functional and gene transcription defects are reversed by activating the DCs with LPS. These results show that dysregulation of MHC-II turnover suppresses DC development and function.

MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4+ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKRKI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKRKI/KI spleen, with the remaining MHC IIKRKI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKRKI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKRKI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKRKI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKRKI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity.

Importance of accessibility to the extracellular juxtamembrane stalk region of membrane protein for substrate recognition by viral ubiquitin ligase K5.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

Activation of Dendritic Cells Alters the Mechanism of MHC Class II Antigen Presentation to CD4 T Cells.

Both immature and mature dendritic cells (DCs) can process and present foreign Ags to CD4 T cells; however, the mechanism by which MHC class II (MHC-II) in mature DCs acquires antigenic peptides remains unknown. To address this, we have studied Ag processing and presentation of two distinct CD4 T cell epitopes of the influenza virus hemagglutinin coat protein by both immature and mature mouse DCs. We find that immature DCs almost exclusively use newly synthesized MHC-II targeted to DM+ late endosomes for presentation to influenza virus-specific CD4 T cells. By contrast, mature DCs exclusively use recycling MHC-II that traffics to both early and late endosomes for antigenic peptide binding. Rab11a knockdown partially inhibits recycling of MHC-II in mature DCs and selectively inhibits presentation of an influenza virus hemagglutinin CD4 T cell epitope generated in early endosomes. These studies highlight a "division of labor" in MHC-II peptide binding, in which immature DCs preferentially present Ags acquired in Rab11a- DM+ late endosomes, whereas mature DCs use recycling MHC-II to present antigenic peptides acquired in both Rab11a+ early endosomes and Rab11a- endosomes for CD4 T cell activation.

Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4ß1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.

MITOL/MARCH5 determines the susceptibility of cardiomyocytes to doxorubicin-induced ferroptosis by regulating GSH homeostasis.

MITOL/MARCH5 is an E3 ubiquitin ligase that plays a crucial role in the control of mitochondrial quality and function. However, the significance of MITOL in cardiomyocytes under physiological and pathological conditions remains unclear. First, to determine the significance of MITOL in unstressed hearts, we assessed the cellular changes with the reduction of MITOL expression by siRNA in neonatal rat primary ventricular cardiomyocytes (NRVMs). MITOL knockdown in NRVMs induced cell death via ferroptosis, a newly defined non-apoptotic programmed cell death, even under no stress conditions. This phenomenon was observed only in NRVMs, not in other cell types. MITOL knockdown markedly reduced mitochondria-localized GPX4, a key enzyme associated with ferroptosis, promoting accumulation of lipid peroxides in mitochondria. In contrast, the activation of GPX4 in MITOL knockdown cells suppressed lipid peroxidation and cell death. MITOL knockdown reduced the glutathione/oxidized glutathione (GSH/GSSG) ratio that regulated GPX4 expression. Indeed, the administration of GSH or N-acetylcysteine improved the expression of GPX4 and viability in MITOL-knockdown NRVMs. MITOL-knockdown increased the expression of the glutathione-degrading enzyme, ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1). The knockdown of Chac1 restored the GSH/GSSG ratio, GPX4 expression, and viability in MITOL-knockdown NRVMs. Further, in cultured cardiomyocytes stressed with DOX, both MITOL and GPX4 were reduced, whereas forced-expression of MITOL suppressed DOX-induced ferroptosis by maintaining GPX4 content. Additionally, MITOL knockdown worsened vulnerability to DOX, which was almost completely rescued by treatment with ferrostatin-1, a ferroptosis inhibitor. In vivo, cardiac-specific depletion of MITOL did not produce obvious abnormality, but enhanced susceptibility to DOX toxicity. Finally, administration of ferrostatin-1 suppressed exacerbation of DOX-induced myocardial damage in MITOL-knockout hearts. The present study demonstrates that MITOL determines the cell fate of cardiomyocytes via the ferroptosis process and plays a key role in regulating vulnerability to DOX treatment. (288/300).

Kaposi's sarcoma-associated herpesvirus ubiquitin ligases downregulate cell surface expression of l-selectin.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi's sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.

Identification of highest neurotoxic amyloid-ß plaque type showing reduced contact with astrocytes.

Amyloid-ß (Aß) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aß plaques determine the pathogenesis of Aß. Here, we categorized Aß plaques into four types based on the macroscopic features of the dense core, and found that the Aß-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aß plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aß plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aß-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aß neuropathology, at least in part, through the reduced contacts between Aß plaques and astrocytes.

Downregulation of MHC Class II by Ubiquitination Is Required for the Migration of CD206+ Dendritic Cells to Skin-Draining Lymph Nodes.

Dendritic cells (DCs) are critical players in skin homeostasis. A subset of mannose receptor (CD206)-expressing monocyte-derived DCs was found in skin, and their migratory counterpart is present in skin-draining lymph nodes (sdLNs). Skin CD206+ DCs were shown to upregulate MHC class II (MHCII) progressively, raising the question of whether this feature affects their biology. In this study, we assessed the role of MHCII regulation in the development and migration of these cells in mouse models expressing differential MHCII levels. Using CD206 as a surrogate marker, we found that skin CD206+ DCs develop in an MHCII-independent manner. However, their migration to sdLNs was affected by overexpression rather than absence or lower expression of MHCII. Accordingly, B16 tumor growth was exacerbated in mice overexpressing MHCII in the absence of ubiquitination. Mechanistically, CD206+ DCs from these mice showed decreased IRF4 and CCR7 expression. LPS, which is known to promote monocyte-derived DC recruitment to sdLNs, partially improved these defects. However, GM-CSF delivery restored CD206+ DC migration by promoting IRF4 expression. Collectively, these data show that MHCII downregulation is crucial for IRF4-dependent migration of CD206+ DCs to sdLNs in health and disease.

MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2.

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells.

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II-peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4(+) T cells.

Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Rα).

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

MHC class II fine tuning by ubiquitination: lesson from MARCHs.

Ubiquitination, a posttranscriptional modification, has been known to contribute to many aspects of cellular event (e.g., protein quality control, signal transduction). In 2007 and 2016, we reported physiological E3 ubiquitin ligases for MHC class II; these are membrane-associated ring-CH-type finger (MARCH)-1 and MARCH-8. Importantly, MARCH-1 and -8 are structurally close to each other, but have different expression profiles. MARCH-1 and -8 are expressed at secondary lymphoid organs and thymic epithelial cells, respectively. These findings suggest contribution of MARCHs to immunological disorders in human; however, its contribution remains to be elucidated. In this review, recent progress on MARCHs will be summarized from molecular and/or immunological point of view and future direction would be discussed.

Inflammasome and Fas-Mediated IL-1ß Contributes to Th17/Th1 Cell Induction in Pathogenic Bacterial Infection In Vivo.

CD4+ Th cells play crucial roles in orchestrating immune responses against pathogenic microbes, after differentiating into effector subsets. Recent research has revealed the importance of IFN-γ and IL-17 double-producing CD4+ Th cells, termed Th17/Th1 cells, in the induction of autoimmune and inflammatory diseases. In addition, Th17/Th1 cells are involved in the regulation of infection caused by the intracellular bacterium Mycobacterium tuberculosis in humans. However, the precise mechanism of Th17/Th1 induction during pathogen infection is unclear. In this study, we showed that the inflammasome and Fas-dependent IL-1ß induces Th17/Th1 cells in mice, in response to infection with the pathogenic intracellular bacterium Listeria monocytogenes In the spleens of infected wild-type mice, Th17/Th1 cells were induced, and expressed T-bet and Rorγt. In Pycard-/- mice, which lack the adaptor molecule of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain), Th17/Th1 induction was abolished. In addition, the Fas-mediated IL-1ß production was required for Th17/Th1 induction during bacterial infection: Th17/Th1 induction was abolished in Fas-/- mice, whereas supplementation with recombinant IL-1ß restored Th17/Th1 induction via IL-1 receptor 1 (IL-1R1), and rescued the mortality of Fas-/- mice infected with Listeria IL-1R1, but not apoptosis-associated speck-like protein containing a caspase recruitment domain or Fas on T cells, was required for Th17/Th1 induction, indicating that IL-1ß stimulates IL-1R1 on T cells for Th17/Th1 induction. These results indicate that IL-1ß, produced by the inflammasome and Fas-dependent mechanisms, contributes cooperatively to the Th17/Th1 induction during bacterial infection. This study provides a deeper understanding of the molecular mechanisms underlying Th17/Th1 induction during pathogenic microbial infections in vivo.

MARCH1 E3 Ubiquitin Ligase Dampens the Innate Inflammatory Response by Modulating Monocyte Functions in Mice.

Ubiquitination was recently identified as a central process in the pathogenesis and development of numerous inflammatory diseases, such as obesity, atherosclerosis, and asthma. Treatment with proteasomal inhibitors led to severe side effects because ubiquitination is heavily involved in a plethora of cellular functions. Thus, new players regulating ubiquitination processes must be identified to improve therapies for inflammatory diseases. In addition to their role in adaptive immunity, endosomal MHC class II (MHCII) molecules were shown to modulate innate immune responses by fine tuning the TLR4 signaling pathway. However, the role of MHCII ubiquitination by membrane associated ring-CH-type finger 1 (MARCH1) E3 ubiquitin ligase in this process remains to be assessed. In this article, we demonstrate that MARCH1 is a key inhibitor of innate inflammation in response to bacterial endotoxins. The higher mortality of March1-/- mice challenged with a lethal dose of LPS was associated with significantly stronger systemic production of proinflammatory cytokines and splenic NK cell activation; however, we did not find evidence that MARCH1 modulates LPS or IL-10 signaling pathways. Instead, the mechanism by which MARCH1 protects against endotoxic shock rests on its capacity to promote the transition of monocytes from Ly6CHi to Ly6C+/- Moreover, in competitive bone marrow chimeras, March1-/- monocytes and polymorphonuclear neutrophils outcompeted wild-type cells with regard to bone marrow egress and homing to peripheral organs. We conclude that MARCH1 exerts MHCII-independent effects that regulate the innate arm of immunity. Thus, MARCH1 might represent a potential new target for emerging therapies based on ubiquitination reactions in inflammatory diseases.

Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells.

MHC class II (MHC-II)-dependent antigen presentation by antigen-presenting cells (APCs) is carefully controlled to achieve specificity of immune responses; the regulated assembly and degradation of antigenic peptide-MHC-II complexes (pMHC-II) is one aspect of such control. In this study, we have examined the role of ubiquitination in regulating pMHC-II biosynthesis, endocytosis, recycling, and turnover in APCs. By using APCs obtained from MHC-II ubiquitination mutant mice, we find that whereas ubiquitination does not affect pMHC-II formation in dendritic cells (DCs), it does promote the subsequent degradation of newly synthesized pMHC-II. Acute activation of DCs or B cells terminates expression of the MHC-II E3 ubiquitin ligase March-I and prevents pMHC-II ubiquitination. Most importantly, this change results in very efficient pMHC-II recycling from the surface of DCs and B cells, thereby preventing targeting of internalized pMHC-II to lysosomes for degradation. Biochemical and functional assays confirmed that pMHC-II turnover is suppressed in MHC-II ubiquitin mutant DCs or by acute activation of wild-type DCs. These studies demonstrate that acute APC activation blocks the ubiquitin-dependent turnover of pMHC-II by promoting efficient pMHC-II recycling and preventing lysosomal targeting of internalized pMHC-II, thereby enhancing pMHC-II stability for efficient antigen presentation to CD4 T cells.

Interleukin 10 (IL-10)-mediated Immunosuppression: MARCH-I INDUCTION REGULATES ANTIGEN PRESENTATION BY MACROPHAGES BUT NOT DENDRITIC CELLS.

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen-specific CD4 T cells; however, the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of antigen presenting cell function in macrophages is March-I-dependent, whereas in DCs, suppression is March- I-independent.

Loss of MHC II ubiquitination inhibits the activation and differentiation of CD4 T cells.

Peptide-MHC class II complexes (pMHC II) are degraded by MARCH-I-mediated ubiquitination, and the stabilization of pMHC II by loss of its ubiquitination is one phenotype defining the activation of conventional dendritic cells (cDCs). However, the role of such stabilization of pMHC II in the context of T-cell activation/differentiation remains unclear. Here, we show that loss of pMHC II ubiquitination inhibits the activation and differentiation of CD4 T cells, probably through down-regulation of CD18/integrin ß2 and their diminished IL-12 production in a cell intrinsic manner. The cDCs generated from mice whose pMHC II ubiquitination is inhibited had a decreased ability to activate naive CD4 T cells and induce Th1/Th17 differentiation. In addition, cDCs whose MHC II ubiquitination was inhibited showed down-regulation of CD18/integrin beta 2 and of IL-12 production. This unexpected finding suggests that loss of MHC II ubiquitination contributes to the negative feedback of CD4 T-cell immune responses.