A membrane-associated MHC-I inhibitory axis for cancer immune evasion.

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.

Pathways of antigen processing.

T cell recognition of antigen-presenting cells depends on their expression of a spectrum of peptides bound to major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I- and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review, we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

The transcription factor TFEB acts as a molecular switch that regulates exogenous antigen-presentation pathways.

Dendritic cells (DCs) can initiate immune responses by presenting exogenous antigens to T cells via both major histocompatibility complex (MHC) class I pathways and MHC class II pathways. Lysosomal activity has an important role in modulating the balance between these two pathways. The transcription factor TFEB regulates lysosomal function by inducing lysosomal activation. Here we report that TFEB expression inhibited the presentation of exogenous antigen by MHC class I while enhancing presentation via MHC class II. TFEB promoted phagosomal acidification and protein degradation. Furthermore, we found that the activation of TFEB was regulated during DC maturation and that phagosomal acidification was impaired in DCs in which the gene encoding TFEB was silenced. Our data indicate that TFEB is a key participant in the differential regulation of the presentation of exogenous antigens by DCs.

Cutting Edge: Phagosome-associated Autophagosomes Containing Antigens and Proteasomes Drive TAP-Independent Cross-Presentation.

Activation of naive CD8-positive T lymphocytes is mediated by dendritic cells that cross-present MHC class I (MHC-I)-associated peptides derived from exogenous Ags. The most accepted mechanism involves the translocation of Ags from phagosomes or endolysosomes into the cytosol, where antigenic peptides generated by cytosolic proteasomes are delivered by the transporter associated with Ag processing (TAP) to the endoplasmic reticulum, or an endocytic Ag-loading compartment, where binding to MHC-I occurs. We have described an alternative pathway where cross-presentation is independent of TAP but remains dependent on proteasomes. We provided evidence that active proteasomes found within the lumen of phagosomes and endolysosomal vesicles locally generate antigenic peptides that can be directly loaded onto trafficking MHC-I molecules. However, the mechanism of active proteasome delivery to the endocytic compartments remained unknown. In this study, we demonstrate that phagosome-associated LC3A/B structures deliver proteasomes into subcellular compartments containing exogenous Ags and that autophagy drives TAP-independent, proteasome-dependent cross-presentation.

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.

SARS-CoV-2 accessory proteins ORF7a and ORF3a use distinct mechanisms to down-regulate MHC-I surface expression.

Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small-protein ß2-microglobulin (ß2m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells, these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8+ T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that down-regulate MHC-I expression to avoid CD8+ T cell recognition. Here, we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, down-regulate MHC-I expression using distinct mechanisms. First, ORF3a, a viroporin, reduces the global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of ß2m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a specific mechanism that allows immune evasion by SARS-CoV-2.

CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

mosGILT controls innate immunity and germ cell development in Anopheles gambiae.

Gene-edited mosquitoes lacking a gamma-interferon-inducible lysosomal thiol reductase-like protein, namely (mosGILTnull) have lower Plasmodium infection, which is linked to impaired ovarian development and immune activation. The transcriptome of mosGILTnull Anopheles gambiae was therefore compared to wild type (WT) mosquitoes by RNA-sequencing to delineate mosGILT-dependent pathways. Compared to WT mosquitoes, mosGILTnull A. gambiae demonstrated altered expression of genes related to oogenesis, 20-hydroxyecdysone synthesis, as well as immune-related genes. Serendipitously, the zero population growth gene, zpg, an essential regulator of germ cell development was found to be one of the most downregulated genes in mosGILTnull mosquitoes. These results provide a crucial missing link between two previous studies on the role of zpg and mosGILT in ovarian development. This study further demonstrates that mosGILT has the potential to serve as a target for the biological control of mosquito vectors and to influence the Plasmodium life cycle within the vector.

Translational shutdown and evasion of the innate immune response by SARS-CoV-2 NSP14 protein.

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14-NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

HLA tapasin independence: broader peptide repertoire and HIV control.

Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.

Intrinsic expression of viperin regulates thermogenesis in adipose tissues.

Viperin is an interferon (IFN)-inducible multifunctional protein. Recent evidence from high-throughput analyses indicates that most IFN-inducible proteins, including viperin, are intrinsically expressed in specific tissues; however, the respective intrinsic functions are unknown. Here we show that the intrinsic expression of viperin regulates adipose tissue thermogenesis, which is known to counter metabolic disease and contribute to the febrile response to pathogen invasion. Viperin knockout mice exhibit increased heat production, resulting in a reduction of fat mass, improvement of high-fat diet (HFD)-induced glucose tolerance, and enhancement of cold tolerance. These thermogenic phenotypes are attributed to an adipocyte-autonomous mechanism that regulates fatty acid ß-oxidation. Under an HFD, viperin expression is increased, and its function is enhanced. Our findings reveal the intrinsic function of viperin as a novel mechanism regulating thermogenesis in adipose tissues, suggesting that viperin represents a molecular target for thermoregulation in clinical contexts.

Impact of Calreticulin and Its Mutants on Endoplasmic Reticulum Function in Health and Disease.

The endoplasmic reticulum (ER) performs key cellular functions including protein synthesis, lipid metabolism and signaling. While these functions are spatially isolated in structurally distinct regions of the ER, there is cross-talk between the pathways. One vital player that is involved in ER function is the ER-resident protein calreticulin (CALR). It is a calcium ion-dependent lectin chaperone that primarily assists in glycoprotein synthesis in the ER as part of the protein quality control machinery. CALR also buffers calcium ion release and mediates other glycan-independent protein interactions. Mutations in CALR have been reported in a subset of chronic blood tumors called myeloproliferative neoplasms. The mutations consist of insertions or deletions in the CALR gene that all cause a + 1 bp shift in the reading frame and lead to a dramatic alteration of the amino acid sequence of the C-terminal domain of CALR. This alters CALR function and affects cell homeostasis. This chapter will discuss how CALR and mutant CALR affect ER health and disease.

Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery.

Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency. We examined a library of poly(amine-co-ester) (PACE) polymers with different end groups using this probe and observed a strong correlation between endosomal escape and transfection efficiency (R2 = 0.9334). In addition, we found that mRNA encapsulation efficiency and endosomal escape, but not uptake, were determinant factors for transfection efficiency. The polymers with high endosomal escape/transfection efficiency in vitro also showed good transfection efficiency in vivo, and mRNA expression was primarily observed in spleens after intravenous delivery. Together, our study suggests that the luciferase probe can be used as an effective tool to quantitate endosomal escape, which is essential for rational optimization of intracellular drug delivery systems.

Cytosolic Processing Governs TAP-Independent Presentation of a Critical Melanoma Antigen.

Cancer immunotherapy has been flourishing in recent years with remarkable clinical success. But as more patients are treated, a shadow is emerging that has haunted other cancer therapies: tumors develop resistance. Resistance is often caused by defects in the MHC class I Ag presentation pathway critical for CD8 T cell-mediated tumor clearance. TAP and tapasin, both key players in the pathway, are frequently downregulated in human cancers, correlating with poor patient survival. Reduced dependence on these factors may promote vaccine efficiency by limiting immune evasion. In this study, we demonstrate that PMEL209-217, a promising phase 3 trial-tested antimelanoma vaccine candidate, is robustly presented by various TAP- and/or tapasin-deficient cell lines. This striking characteristic may underlie its potency as a vaccine. Surprisingly, cytosolic proteasomes generate the peptide even for TAP-independent presentation, whereas tripeptidyl peptidase 2 (TPP2) efficiently degrades the epitope. Consequently, inhibiting TPP2 substantially boosts PMEL209-217 presentation, suggesting a possible strategy to improve the therapeutic efficacy of the vaccine.

Structural studies of viperin, an antiviral radical SAM enzyme.

Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (ßα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first ß-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export.

Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8+ T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and ß2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.