The protease DDI2 regulates NRF1 activation in response to cadmium toxicity.

DNA-damage inducible 1 homolog 2 (DDI2) is a protease that activates the transcription factor NRF1. Cellular models have shown that this pathway contributes to cell-stress adaptation, for example, on proteasome inhibition. However, DDI2 physiological function is unknown. Ddi2 Knock-out (KO) mice were embryonic lethal. Therefore, we generated liver-specific Ddi2-KO animals and used comprehensive genetic analysis to identify the molecular pathways regulated by DDI2. Here, we demonstrate that DDI2 contributes to metallothionein (MT) expression in mouse and human hepatocytes at basal and upon cadmium (Cd) exposure. This transcriptional program is dependent on DDI2-mediated NRF1 proteolytic maturation. In contrast, NRF1 homolog NRF2 does not contribute to MT production. Mechanistically, we observed that Cd exposure inhibits proteasome activity, resulting in DDI2-mediated NRF1 proteolytic maturation. In line with these findings, DDI2 deficiency sensitizes cells to Cd toxicity. This study identifies a function for DDI2 that links proteasome homeostasis to heavy metal mediated toxicity.

The aspartyl protease DDI2 drives adaptation to proteasome inhibition in multiple myeloma.

Proteasome inhibitors, such as bortezomib, are first-line therapy against multiple myeloma (MM). Unfortunately, patients frequently become refractory to this treatment. The transcription factor NRF1 has been proposed to initiate an adaptation program that regulates proteasome levels. In the context of proteasome inhibition, the cytosolic protease DDI2 cleaves NRF1 to release an active fragment that translocates to the nucleus to promote the transcription of new proteasome subunits. However, the contribution of the DDI2-NRF1 pathway to bortezomib resistance is poorly understood. Here we show that upon prolonged bortezomib treatment, MM cells become resistant to proteasome inhibition by increasing the expression of DDI2 and consequently activation of NRF1. Furthermore, we found that many MM cells became more sensitive to proteasome impairment in the context of DDI2 deficiency. Mechanistically, we demonstrate that both the protease and the HDD domains of DDI2 are required to activate NRF1. Finally, we show that partial inhibition of the DDI2-protease domain with the antiviral drug nelfinavir increased bortezomib susceptibility in treated MM cells. Altogether, these findings define the DDI2-NRF1 pathway as an essential program contributing to proteasome inhibition responses and identifying DDI2 domains that could be targets of interest in bortezomib-treated MM patients.

Development of ICT01, a first-in-class, anti-BTN3A antibody for activating Vγ9Vδ2 T cell-mediated antitumor immune response.

Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti­butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen­major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)­binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αß T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.

BTN2A1, an immune checkpoint targeting Vγ9Vδ2 T cell cytotoxicity against malignant cells.

The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

MARCH9-mediated ubiquitination regulates MHC I export from the TGN.

Given the heterogeneous nature of antigens, major histocompatibility complex class I (MHC I) intracellular transport intersects with multiple degradation pathways for efficient peptide loading and presentation to cytotoxic T cells. MHC I loading with peptides in the endoplasmic reticulum (ER) is a tightly regulated process, while post-ER intracellular transport is considered to occur by default, leading to peptide-bearing MHC I delivery to the plasma membrane. We show here that MHC I traffic is submitted to a previously uncharacterized sorting step at the trans Golgi network (TGN), dependent on the ubiquitination of its cytoplasmic tail lysine residues. MHC I ubiquitination is mediated by the E3 ligase membrane-associated RING-CH 9 (MARCH9) and allows MHC I access to Syntaxin 6-positive endosomal compartments. We further show that MARCH9 can also target the human MHC I-like lipid antigen-presentation molecule CD1a. MARCH9 expression is modulated by microbial pattern exposure in dendritic cells (DCs), thus revealing the role of this ubiquitin E3 ligase in coordinating MHC I access to endosomes and DC activation for efficient antigen cross-presentation.

Impairment of both IRE1 expression and XBP1 activation is a hallmark of GCB DLBCL and contributes to tumor growth.

The endoplasmic reticulum kinase inositol-requiring enzyme 1 (IRE1) and its downstream target X-box-binding protein 1 (XBP1) drive B-cell differentiation toward plasma cells and have been shown to contribute to multiple myeloma development; yet, little is known of the role of this pathway in diffuse large B-cell lymphoma (DLBCL). Here, we show that in the germinal center B-cell-like (GCB) DLBCL subtype, IRE1 expression is reduced to a level that prevents XBP1 activation. Gene expression profiles indicated that, in GCB DLBCL cancer samples, expression of IRE1 messenger RNA was inversely correlated with the levels and activity of the epigenetic repressor, histone methyltransferase enhancer of zeste homolog 2 (EZH2). Correspondingly, in GCB-derived cell lines, the IRE1 promoter carried increased levels of the repressive epigenetic mark histone 3 lysine 27 trimethylation. Pharmacological inhibition of EZH2 erased those marks and restored IRE1 expression and function in vitro and in vivo. Moreover, reconstitution of the IRE1-signaling pathway, by expression of the XBP1-active form, compromised GCB DLBCL tumor growth in a mouse xenograft cancer model. These findings indicate that IRE1-XBP1 downregulation distinguishes GCB DLBCL from other DLBCL subtypes and contributes to tumor growth.

Pharmacological eEF2K activation promotes cell death and inhibits cancer progression.

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV-PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti-viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir-resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir-mediated anti-tumoral activity is severely compromised in eEF2K-deficient engrafted tumors in vivo Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti-tumoral properties of HIV-PIs.

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity.

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.

T Cell Priming by Activated Nlrc5-Deficient Dendritic Cells Is Unaffected despite Partially Reduced MHC Class I Levels.

NLRC5, a member of the NOD-like receptor (NLR) protein family, has recently been characterized as the master transcriptional regulator of MHCI molecules in lymphocytes, in which it is highly expressed. However, its role in activated dendritic cells (DCs), which are instrumental to initiate T cell responses, remained elusive. We show in this study that, following stimulation of DCs with inflammatory stimuli, not only did NLRC5 level increase, but also its importance in directing MHCI transcription. Despite markedly reduced mRNA and intracellular H2-K levels, we unexpectedly observed nearly normal H2-K surface display in Nlrc5(-/-) DCs. Importantly, this discrepancy between a strong intracellular and a mild surface defect in H2-K levels was observed also in DCs with H2-K transcription defects independent of Nlrc5. Hence, alongside with demonstrating the importance of NLRC5 in MHCI transcription in activated DCs, we uncover a general mechanism counteracting low MHCI surface expression. In agreement with the decreased amount of neosynthesized MHCI, Nlrc5(-/-) DCs exhibited a defective capacity to display endogenous Ags. However, neither T cell priming by endogenous Ags nor cross-priming ability was substantially affected in activated Nlrc5(-/-) DCs. Altogether, these data show that Nlrc5 deficiency, despite significantly affecting MHCI transcription and Ag display, is not sufficient to hinder T cell activation, underlining the robustness of the T cell priming process by activated DCs.

An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response.

Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.

Pyroptosis: Caspase-11 Unlocks the Gates of Death.

How inflammatory caspases trigger pyroptotic cell death is mostly unexplained. In this issue of Immunity, Núñez and colleagues report that caspase-11 cleaves the transmembrane channel pannexin-1, causing an efflux of cellular ATP that promotes a P2X7 receptor-dependent pyroptosis.

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

MHC-II ubiquitination.

Ubiquitinated protein detection is often troublesome since in most cases this modification reduces the half-life of targeted proteins, inducing their degradation. Furthermore, ubiquitination is reversible thanks to the action of highly specific deubiquitinases present in all eukaryotic cells. MHC molecules ubiquitination has been demonstrated to be a key event in the regulation of the potent immunostimulatory properties of activated human dendritic cells.

Autophagy inhibition promotes defective neosynthesized proteins storage in ALIS, and induces redirection toward proteasome processing and MHCI-restricted presentation.

A significant portion of newly synthesized protein fails to fold properly and is quickly degraded. These defective ribosomal products (DRiPs) are substrates for the ubiquitin-proteasome system (UPS) and give rise to a large fraction of peptides presented by major histocompatibility complex class I molecules (MHCI). Here, we showed that DRiPs are also autophagy substrates, which accumulate upon autophagy inhibition in aggresome-like-induced structures (ALIS). Aggregation is critically depending on p62/SQSTM1, but occurs in the absence of activation of the NRF2 signaling axis and transcriptional regulation of p62/SQSTM1. We demonstrated that autophagy-targeted DRiPs can become UPS substrates and give rise to MHCI presented peptides upon autophagy inhibition. We further demonstrated that autophagy targeting of DRiPs is controlled by NBR1, but not p62/SQSTM1, CHIP or BAG-1. Active autophagy therefore directly modulates MHCI presentation by constantly degrading endogenous defective neosynthesized antigens, which are submitted to at least two distinct quality control mechanisms.

Tuning of natural killer cell reactivity by NKp46 and Helios calibrates T cell responses.

Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46. The down-regulation of NK cell activity by NKp46 was associated with the silencing of the Helios transcription factor in NK cells. NKp46 was critical for the subsequent development of antiviral and antibacterial T cell responses, which suggests that the regulation of NK cell function by NKp46 allows for the optimal development of adaptive immune responses. NKp46 blockade enhanced NK cell reactivity in vivo, which could enable the design of immunostimulation strategies in humans.

BAD-LAMP is a novel biomarker of nonactivated human plasmacytoid dendritic cells.

The brain and dendritic cell (BAD)-associated lysosome-associated membrane protein (LAMP)-like molecule (BAD-LAMP, c20orf103, UNC-46) is a newly identified member of the family of LAMPs. BAD-LAMP expression in the mouse is confined to neurons. We demonstrate here that in humans, BAD-LAMP can specifically be found in the type I IFN-producing plasmacytoid dendritic cells (pDCs). Human BAD-LAMP is localized in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of freshly isolated CD123(+) pDCs and is rapidly lost upon activation by unmethylated cytosine-phosphate-guanine (CpG) oligonucleotides. The restricted pattern of BAD-LAMP expression allows for the rapid identification of normal and leukemic human pDCs in tissues and blood.

TLR8 deficiency leads to autoimmunity in mice.

TLRs play an essential role in the induction of immune responses by detecting conserved molecular products of microorganisms. However, the function of TLR8 is largely unknown. In the current study, we investigated the role of TLR8 signaling in immunity in mice. We found that Tlr8(-/-) DCs overexpressed TLR7, were hyperresponsive to various TLR7 ligands, and showed stronger and faster NF-κB activation upon stimulation with the TLR7 ligand R848. Tlr8(-/-) mice showed splenomegaly, defective development of marginal zone (MZ) and B1 B cells, and increased serum levels of IgM and IgG2a. Furthermore, Tlr8(-/-) mice exhibited increased serum levels of autoantibodies against small nuclear ribonucleoproteins, ribonucleoprotein, and dsDNA and developed glomerulonephritis, whereas neither Tlr7(-/-) nor Tlr8(-/-)Tlr7(-/-) mice showed any of the phenotypes observed in Tlr8(-/-) mice. These data provide evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7 expression and prevention of spontaneous autoimmunity.

Ribosomal protein mRNAs are translationally-regulated during human dendritic cells activation by LPS.

BACKGROUND: Dendritic cells (DCs) are the sentinels of the mammalian immune system, characterized by a complex maturation process driven by pathogen detection. Although multiple studies have described the analysis of activated DCs by transcriptional profiling, recent findings indicate that mRNAs are also regulated at the translational level. A systematic analysis of the mRNAs being translationally regulated at various stages of DC activation was performed using translational profiling, which combines sucrose gradient fractionation of polysomal-bound mRNAs with DNA microarray analysis. RESULTS: Total and polysomal-bound mRNA populations purified from immature, 4 h and 16 h LPS-stimulated human monocyte-derived DCs were analyzed on Affymetrix microarrays U133 2.0. A group of 375 transcripts was identified as translationally regulated during DC-activation. In addition to several biochemical pathways related to immunity, the most statistically relevant biological function identified among the translationally regulated mRNAs was protein biosynthesis itself. We singled-out a cluster of 11 large ribosome proteins mRNAs, which are disengaged from polysomes at late time of maturation, suggesting the existence of a negative feedback loop regulating translation in DCs and linking ribosomal proteins to immuno-modulatory function. CONCLUSION: Our observations highlight the importance of translation regulation during the immune response, and may favor the identification of novel protein networks relevant for immunity. Our study also provides information on the potential absence of correlation between gene expression and protein production for specific mRNA molecules present in DCs.

Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein.

Exosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM-BLV-CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM-BLV-CD8-containing exosomes are likely formed from a recycling endosomal/TGN compartment.