Variations in MHC Class II Antigen Processing and Presentation in Health and Disease.

MHC class II (MHC-II) molecules are critical in the control of many immune responses. They are also involved in most autoimmune diseases and other pathologies. Here, we describe the biology of MHC-II and MHC-II variations that affect immune responses. We discuss the classic cell biology of MHC-II and various perturbations. Proteolysis is a major process in the biology of MHC-II, and we describe the various components forming and controlling this endosomal proteolytic machinery. This process ultimately determines the MHC-II-presented peptidome, including cryptic peptides, modified peptides, and other peptides that are relevant in autoimmune responses. MHC-II also variable in expression, glycosylation, and turnover. We illustrate that MHC-II is variable not only in amino acids (polymorphic) but also in its biology, with consequences for both health and disease.

An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport.

Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

RNF26 binds perinuclear vimentin filaments to integrate ER and endolysosomal responses to proteotoxic stress.

Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear. Here, we identify vimentin intermediate filaments as perinuclear anchors for the ER and endolysosomes. We show that perinuclear vimentin filaments engage the ER-embedded RING finger protein 26 (RNF26) at the C-terminus of its RING domain. This restricts RNF26 to perinuclear ER subdomains and enables the corresponding spatial retention of endolysosomes through RNF26-mediated membrane contact sites (MCS). We find that both RNF26 and vimentin are required for the perinuclear coalescence of the ERQC and its juxtaposition with proteolytic compartments, which facilitates efficient recovery from ER stress via the Sec62-mediated ER-phagy pathway. Collectively, our findings reveal a scaffolding mechanism that underpins the spatiotemporal integration of organelles during cellular proteostasis.

MED12 controls the response to multiple cancer drugs through regulation of TGF-ß receptor signaling.

Inhibitors of the ALK and EGF receptor tyrosine kinases provoke dramatic but short-lived responses in lung cancers harboring EML4-ALK translocations or activating mutations of EGFR, respectively. We used a large-scale RNAi screen to identify MED12, a component of the transcriptional MEDIATOR complex that is mutated in cancers, as a determinant of response to ALK and EGFR inhibitors. MED12 is in part cytoplasmic where it negatively regulates TGF-ßR2 through physical interaction. MED12 suppression therefore results in activation of TGF-ßR signaling, which is both necessary and sufficient for drug resistance. TGF-ß signaling causes MEK/ERK activation, and consequently MED12 suppression also confers resistance to MEK and BRAF inhibitors in other cancers. MED12 loss induces an EMT-like phenotype, which is associated with chemotherapy resistance in colon cancer patients and to gefitinib in lung cancer. Inhibition of TGF-ßR signaling restores drug responsiveness in MED12(KD) cells, suggesting a strategy to treat drug-resistant tumors that have lost MED12.

Evolutionarily conserved amino acids in MHC-II mediate bat influenza A virus entry into human cells.

The viral hemagglutinins of conventional influenza A viruses (IAVs) bind to sialylated glycans on host cell surfaces for attachment and subsequent infection. In contrast, hemagglutinins of bat-derived IAVs target major histocompatibility complex class II (MHC-II) for cell entry. MHC-II proteins from various vertebrate species can facilitate infection with the bat IAV H18N11. Yet, it has been difficult to biochemically determine the H18:MHC-II binding. Here, we followed a different approach and generated MHC-II chimeras from the human leukocyte antigen DR (HLA-DR), which supports H18-mediated entry, and the nonclassical MHC-II molecule HLA-DM, which does not. In this context, viral entry was supported only by a chimera containing the HLA-DR α1, α2, and ß1 domains. Subsequent modeling of the H18:HLA-DR interaction identified the α2 domain as central for this interaction. Further mutational analyses revealed highly conserved amino acids within loop 4 (N149) and ß-sheet 6 (V190) of the α2 domain as critical for virus entry. This suggests that conserved residues in the α1, α2, and ß1 domains of MHC-II mediate H18-binding and virus propagation. The conservation of MHC-II amino acids, which are critical for H18N11 binding, may explain the broad species specificity of this virus.

Balanced Epigenetic Regulation of MHC Class I Expression in Tumor Cells by the Histone Ubiquitin Modifiers BAP1 and PCGF1.

MHC class I (MHC-I) molecules are critical for CD8+ T cell responses to viral infections and malignant cells, and tumors can downregulate MHC-I expression to promote immune evasion. In this study, using a genome-wide CRISPR screen on a human melanoma cell line, we identified the polycomb repressive complex 1 (PRC1) subunit PCGF1 and the deubiquitinating enzyme BAP1 as opposite regulators of MHC-I transcription. PCGF1 facilitates deposition of ubiquitin at H2AK119 at the MHC-I promoters to silence MHC-I, whereas BAP1 removes this modification to restore MHC-I expression. PCGF1 is widely expressed in tumors and its depletion increased MHC-I expression in multiple tumor lines, including MHC-Ilow tumors. In cells characterized by poor MHC-I expression, PRC1 and PRC2 act in parallel to impinge low transcription. However, PCGF1 depletion was sufficient to increase MHC-I expression and restore T cell-mediated killing of the tumor cells. Taken together, our data provide an additional layer of regulation of MHC-I expression in tumors: epigenetic silencing by PRC1 subunit PCGF1.

A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation.

MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFß. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

Epstein-Barr virus nuclear antigen EBNA3A modulates IRF3-dependent IFNß expression.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNß induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNß transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor interferon regulatory factor 3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits the binding of interferon regulatory factor 3 to the IFNß promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A.

Choreographing the motor-driven endosomal dance.

The endosomal system orchestrates the transport of lipids, proteins and nutrients across the entire cell. Along their journey, endosomes mature, change shape via fusion and fission, and communicate with other organelles. This intriguing endosomal choreography, which includes bidirectional and stop-and-go motions, is coordinated by the microtubule-based motor proteins dynein and kinesin. These motors bridge various endosomal subtypes to the microtubule tracks thanks to their cargo-binding domain interacting with endosome-associated proteins, and their motor domain interacting with microtubules and associated proteins. Together, these interactions determine the mobility of different endosomal structures. In this Review, we provide a comprehensive overview of the factors regulating the different interactions to tune the fascinating dance of endosomes along microtubules.

Ubiquitin and its relatives as wizards of the endolysosomal system.

The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub- and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells.

The Immunoproteasome Cleans up after Inflammation.

Immune cells and cells activated by the inflammatory cytokine interferon express variant proteasomes called immunoproteasomes that are characterized by unique catalytic subunits. Seifert et al. (2010) now show in mouse models of inflammatory disease that immunoproteasomes help prevent the accumulation of harmful protein aggregates.

Diversifying the anthracycline class of anti-cancer drugs identifies aclarubicin for superior survival of acute myeloid leukemia patients.

The efficacy of anthracycline-based chemotherapeutics, which include doxorubicin and its structural relatives daunorubicin and idarubicin, remains almost unmatched in oncology, despite a side effect profile including cumulative dose-dependent cardiotoxicity, therapy-related malignancies and infertility. Detoxifying anthracyclines while preserving their anti-neoplastic effects is arguably a major unmet need in modern oncology, as cardiovascular complications that limit anti-cancer treatment are a leading cause of morbidity and mortality among the 17 million cancer survivors in the U.S. In this study, we examined different clinically relevant anthracycline drugs for a series of features including mode of action (chromatin and DNA damage), bio-distribution, anti-tumor efficacy and cardiotoxicity in pre-clinical models and patients. The different anthracycline drugs have surprisingly individual efficacy and toxicity profiles. In particular, aclarubicin stands out in pre-clinical models and clinical studies, as it potently kills cancer cells, lacks cardiotoxicity, and can be safely administered even after the maximum cumulative dose of either doxorubicin or idarubicin has been reached. Retrospective analysis of aclarubicin used as second-line treatment for relapsed/refractory AML patients showed survival effects similar to its use in first line, leading to a notable 23% increase in 5-year overall survival compared to other intensive chemotherapies. Considering individual anthracyclines as distinct entities unveils new treatment options, such as the identification of aclarubicin, which significantly improves the survival outcomes of AML patients while mitigating the treatment-limiting side-effects. Building upon these findings, an international multicenter Phase III prospective study is prepared, to integrate aclarubicin into the treatment of relapsed/refractory AML patients.

SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport.

The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or "late" endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles.

Monitoring Anthracycline Cancer Drug-Nucleosome Interaction by NMR Using a Specific Isotope Labeling Approach for Nucleosomal DNA.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding to the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption.

Internal circadian misallignment in a mouse model of chemotherapy induced fatigue.

BACKGROUND: Cancer survivors can experience long lasting fatigue resulting in a lower quality of life. How chemotherapy treatment contributes to this fatigue is poorly understood. Previously we have shown in a mouse model of cancer related fatigue that doxorubicin treatment induces fatigue-like symptoms related to disturbed circadian rhythms. However, the specific components of the circadian regulatory circuitry affected by doxorubicin treatment remained unclear. Therefore we investigated the role of the central circadian clock, the suprachiasmatic nucleus (SCN), in chemotherapy-induced fatigue. METHODS: We measured circadian controlled behavior and multiunit neuronal activity in the SCN in freely moving mice exhibiting fatigue-like behavior after doxorubicin treatment under both light-dark (LD) and constant dark (DD) conditions. Additionally, we assessed the expression of inflammation related genes in spleen and kidney as potential inducers of CRF. RESULTS: Doxorubicin treatment significantly reduced both the running wheel activity and time spent using the running wheel for over five weeks after treatment. In contrast to the pronounced effects on behavior and neuronal activity of doxorubicin on circadian rhythms, peripheral inflammation markers only showed minor differences, five weeks after the last treatment. Surprisingly, the circadian SCN neuronal activity under both LD and DD conditions was not affected. However, the circadian timing of neuronal activity in peri-SCN areas (the brain areas surrounding SCN) and circadian rest-activity behavior was strongly affected by doxorubicin, suggesting that the output of the SCN was altered. The reduced correlation between the SCN neuronal activity and behavioral activity after doxorubicin treatment, suggests that the information flow from the SCN to the periphery was disturbed. CONCLUSION: Our preclinical study suggests that chemotherapy-induced fatigue disrupts the circadian rhythms in peripheral brain areas and behavior downstream from the SCN, potentially leading to fatigue like symptoms. Our data suggest that peripheral inflammation responses are less important for the maintenance of fatigue. Chronotherapy that realigns circadian rhythms could represent a non-invasive way to improve patient outcomes following chemotherapy.

Complement is a central mediator of radiotherapy-induced tumor-specific immunity and clinical response.

Radiotherapy induces DNA damage and cell death, but recent data suggest that concomitant immune stimulation is an integral part of the therapeutic action of ionizing radiation. It is poorly understood how radiotherapy supports tumor-specific immunity. Here we report that radiotherapy induced tumor cell death and transiently activated complement both in murine and human tumors. The local production of pro-inflammatory anaphylatoxins C3a and C5a was crucial to the tumor response to radiotherapy and concomitant stimulation of tumor-specific immunity. Dexamethasone, a drug frequently given during radiotherapy, limited complement activation and the anti-tumor effects of the immune system. Overall, our findings indicate that anaphylatoxins are key players in radiotherapy-induced tumor-specific immunity and the ensuing clinical responses.

CRISPR Activation Screening Identifies VGLL3-TEAD1-RUNX1/3 as a Transcriptional Complex for PD-L1 Expression.

The PD-L1/2-PD-1 immune checkpoint is essential for the proper induction of peripheral tolerance and limits autoimmunity, whereas tumor cells exploit their expression to promote immune evasion. Many different cell types express PD-L1/2, either constitutively or upon stimulation, but the factors driving this expression are often poorly defined. In this study, using genome-wide CRISPR activation screening, we identified three factors that upregulate PD-L1 expression: GATA2, MBD6, and transcription cofactor vestigial-like protein 3 (VGLL3). VGLL3 acts as a transcriptional regulator, and its expression induced PD-L1 in many different cell types. Conversely, loss of VGLL3 impaired IFN-γ-induced PD-L1/2 expression in human keratinocytes. Mechanistically, by performing a second screen to identify proteins acting in concert with VGLL3, we found that VGLL3 forms a complex with TEAD1 and RUNX1/3 to drive expression of PD-L1/2. Collectively, our work identified a new transcriptional complex controlling PD-L1/2 expression and suggests that VGLL3, in addition to its known role in the expression of proinflammatory genes, can balance inflammation by upregulating the anti-inflammatory factors PD-L1 and PD-L2.