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.
How inflammatory caspases trigger pyroptotic cell death is mostly unexplained. Nunez 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.
Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1β. 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.
In response to Toll-like receptor ligands, dendritic cells (DCs) dramatically enhance their antigen presentation capacity by stabilizing at the cell-surface MHC II molecules. We demonstrate here ...that, in human monocyte-derived DCs, the RING-CH ubiquitin E3 ligase, membrane-associated RING-CH I (MARCH I), promotes the ubiquitination of the HLA-DR β-chain. Thus, in nonactivated DCs, MARCH I induces the surface internalization of mature HLA-DR complexes, therefore reducing their stability and levels. We further demonstrate that the maturation-dependent down-regulation of MARCH I is a key event in MHC class II up-regulation at the surface of LPS-activated DCs. MARCH I is, therefore, a major regulator of HLA-DR traffic, and its loss contributes to the acquisition of the potent immunostimulatory properties of mature human DCs.
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.
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.
TLR8 deficiency leads to autoimmunity in mice Demaria, Olivier; Pagni, Philippe P; Traub, Stephanie ...
The Journal of clinical investigation,
10/2010, Volume:
120, Issue:
10
Journal Article
Peer reviewed
Open access
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.
IL‐10 is a potent anti‐inflammatory cytokine interfering with antigen presentation by inducing the intracellular sequestration of MHC class II (MHC‐II) molecules. Here we studied the contribution of ...membrane‐associated RING‐CH (MARCH) ubiquitin ligase family members to the IL‐10‐induced down‐regulation of MHC‐II molecules. We found that MARCH1 and MARCH8 proteins are the most potent family members for the down‐regulation of MHC‐II surface expression in transfected cells, but only MARCH1 mRNA expression is strongly induced by IL‐10 in human primary monocytes. We detected mono‐ and poly‐ubiquitinated forms of MHC‐II molecules both in IL‐10‐treated monocytes and in cells transfected with MARCH1. We also show direct interaction between MHC‐II and MARCH1 molecules in co‐immunoprecipitation assays. Finally, we found that siRNA‐mediated knockdown of MARCH1 reverses IL‐10‐induced MHC‐II down‐regulation in primary monocytes. Thus, the immunosuppressive effect of IL‐10 on antigen presentation is mediated through induced expression of MARCH1.
BackgroundICT01 is a first-in-class anti-BTN3A mAb that selectively activates γ9δ2 T cells, leading to remodeling of the tumor microenvironment by activated γ9δ2 T, CD8 T, and NK cells ...(EVICTION-NCT04243499). However, many cancer patients have very low circulating γ9δ2 T cells that limit their response to ICT01. In the EVICTION-2 trial, a novel regimen of ICT01 plus low dose subcutaneous (LDSC) IL-2 is being investigated in patients with advanced-stage solid tumors to increase the number of γ9δ2 T cells that generate a more efficacious anti-tumor immune response.MethodsICT01 (1, 5, 20 or 75 mg, IV Q3W) is given in combination with IL-2 (Proleukin®, 1 or 2 MIU/m2, SC) on days 1–5 of the first 3 cycles and will be continued alone thereafter. Per dose combination two patients are enrolled for dose escalation based on the BOIN simulations to identify a dose regimen that safely expands γ9δ2 T cells, which will be expanded to 6 pts for recommended phase 2 dose regimens. The study received ethics approval for all sites involved.ResultsNineteen patients have completed at least one cycle of ICT01 plus IL-2. Treatment-related adverse events were mainly mild to moderate infusion-related reactions, comparable to those observed with ICT01 or IL-2 monotherapy. No dose-limiting toxicities were reported. A 2–11x increase of γ9δ2 T cell counts above baseline was observed for all 3 cycles across all cohorts peaking around day 8 to 15, which appeared greater at low doses where ICT01 was rapidly cleared, while prolonged target occupancy/activation by high doses prevented γ9δ2 T cells from remaining in the circulation. Activation, mobilization, and proliferation of CD8 T cells (2–3x) and NK cells (2–9x) cycles was similarly observed. Elevated levels of IFNγ, TNFα, IL-6 and IL-8 peaked at ~4 hours post ICT01/IL-2 dose that returned to baseline despite expansion of γ9δ2 T cells. Increased Tregs by flow cytometry appear to be greater at lower doses of ICT01, which may be similar to effects observed in NHPs. Response data by RECIST1.1 every 8 weeks and IHC of tumor biopsies collected at baseline and on Day 28 will be presented.ConclusionICT01 plus LDSC IL-2 produces a broad anti-tumor immune response that is durable across multiple treatment cycles, which appears different to prior attempts to expand g9d2 T cells with bisphosphonates or phosphoantigens.
BackgroundICT01, a novel, anti-BTN3A immunotherapeutic mAb for activating g9d2T cells, is currently evaluated in a Phase 1/2a clinical trial in patients with advanced-stage, relapsed/refractory ...cancer (NCT04243499, EVICTION). ICT01 indirectly activates g9d2 T cells that secrete inflammatory cytokines and migrate into tumors to coordinate antitumor immune responses. Therefore, the baseline number of g9d2 T effector cells constitutes a biomarker of interest and a potential selection criterion for target patients.MethodsFull immunophenotyping (cell counts and activation state) was performed by flow cytometry on fresh blood collected pre- and on-treatment. Serum cytokines were monitored at baseline and post-treatment. Tumor biopsies were harvested at baseline and on Day 28, and multiplex IHC coupled with digital pathology was used to quantify g9d2T cell, CD8 T cell, NK cell, and T reg infiltration and activation stateResultsBaseline circulating g9d2 T cell count was highly variable in solid tumor patients enrolled in the monotherapy arm of EVICTION (median 6918 cell/mL, n=26). Melanoma and colorectal patients displayed respectively the highest (median 42277 cell/mL, n=3) and the lowest (median 3040 cell/mL, n=9) baseline number. During the dose escalation phase, g9d2 T cell activation (CD69+) and migration from the blood was observed 30 min post-ICT01 administration. Serum cytokine levels showed variability within ICT01 dose cohorts. IFNg, TNFa, IL-6 and IL-8 levels post-ICT01 dosing were ICT01 dose dependent and clearly related to baseline number of circulating g9d2 T cells. Activation of peripheral blood NK cells, granulocytes and CD8 T cells was observed post-dosing at ICT01 doses ≥7 mg, which was significantly correlated with baseline g9d2 T cell counts, but not with other immune subsets (Spearman r=0.51, 0.47 and 0.65 for CD69+NK, CD69+CD8 and PD-L1+granulocytes respectively, p<0.05, n=19). Baseline circulating g9d2 T cell count was positively correlated with gdTCR+ T cell density in baseline tumor biopsies (Spearman r=0.76, p=0.0086, n=11). Finally, a trend was observed between baseline g9d2 T cell counts and overall tumor immune cell infiltration and activation post-ICT01 treatment, with 4 patients (out of 13 with available biopsy pairs) with g9d2 T cell counts above the median displaying the highest tumor immune cell infiltration and activation.ConclusionsThese results suggest the utility of measuring baseline g9d2 T cells as part of the patient selection process for ICT01 clinical trials. Patient enrichment based on this biomarker will be tested in EVICTION expansion arms where a minimum baseline threshold of g9d2 T cells counts will be one of the eligibility criteria.Trial RegistrationNCT04243499Ethics ApprovalThe study has obtained Competent Authority and Ethics Committee approvals. Informed consent forms were obtained from all enrolled patients.
Backgroundγ9δ2 T-cells are attractive mediators of cancer immunotherapy due to their strong cytolytic and pro-inflammatory activities and the positive correlation between tumor infiltration and good ...prognosis 1,2. ICT01, a novel anti-BTN3A mAb activating γ9δ2 T-cells, is being evaluated in a Phase 1/2a clinical study (NCT04243499)3,4. Previous studies have shown that IL-2 (Proleukin®) promotes γ9δ2 T-cells expansion following ICT01 stimulation, which may be clinically useful given that γ9δ2 T-cells are normally <5% of total T-cells 5. However, the severe toxicity of IL-2 has limited its widespread use. NL-201 is a de novo alpha-independent IL-2/IL-15 agonist that preferentially stimulates CD8 T and NK cell proliferation at low concentrations, enabling a potentially wider therapeutic index than IL-2, and is being evaluated in a Phase 1 clinical study (NCT04659629)6,7. Here, we explore the potential of ICT01 and NL-201 to synergistically stimulate the activation and proliferation of γ9δ2 T-cells.MethodsFlow cytometry was used to assess IL-2R signaling (pSTAT5), and γ9δ2 T-cell activation and expansion after in vitro culture of huPBMCs with ICT01, NL201 or the combination. Tumor cell killing activity was monitored upon co-culture of huPBMCs with tumor cell lines (Incucyte). In vivo pharmacology was performed in NCG mice engrafted with 20x106 huPBMCs and treated with ICT01 (1 mg/kg IV)±NL-201 (1, 3 or 10 µg/kg IV). Immune cells were phenotyped by flow cytometry in blood and organs collected at sacrifice (Day 16).ResultsNL-201 is ~100X more potent than IL-2 in triggering IL-2R signaling in γ9δ2 T-cells, without preferential activity on Tregs. NL-201 plus ICT01 induces synergistic expansion of γ9δ2 T-cells, approaching ~50% of T-cells after 8 days versus ~10% with single agents. In addition, the combination of NL-201 and ICT01 promotes γ9δ2 T-cell effector memory differentiation, in contrast to IL-2, which induces primarily central memory phenotype. Importantly, NL-201 enhances ICT01-mediated killing of cancer cells by γ9δ2 T-cells.In mice, a dose-dependent expansion of peripheral γ9δ2 T-cells from ~1–2% at baseline to up to 40% of T-cells was observed in the ICT01+NL-201 combination groups. Consistently, γ9δ2 T-cell number and frequency increase in spleen and lungs of the ICT01+NL-201 treated animals as compared to controls. Expanded γ9δ2 T-cells in the combination groups display an effector memory phenotype, confirming our in vitro results.ConclusionsThese results demonstrate the ability of the ICT01+NL-201 combination to synergistically trigger γ9δ2 T-cell activation, expansion and anti-tumor activity and support clinical evaluation of this combination as a novel therapeutic approach for cancer patients.ReferencesGentles, A. J. et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med 21, 938-945, doi:10.1038/nm.3909 (2015).Tosolini, M. et al. Assessment of tumor-infiltrating TCRVgamma9Vdelta2 gammadelta lymphocyte abundance by deconvolution of human cancers microarrays. Oncoimmunology 6, e1284723, doi:10.1080/2162402X.2017.1284723 (2017).Gassart, A. d. et al. 687 Enhancement of anti-tumor immunity by ICT01: a novel g9d2 T cell-activating antibody targeting butyrophilin-3A (BTN3A). Journal for ImmunoTherapy of Cancer 8, A412-A413, doi:10.1136/jitc-2020-SITC2020.0687 (2020).Marabelle, A. et al. 316 EVICTION Study: Preliminary results in solid tumor patients with ICT01, a first-in-class, gamma9 delta2 T cell activating antibody targeting butyrophilin-3A. Journal for ImmunoTherapy of Cancer 8, A194-A195, doi:10.1136/jitc-2020-SITC2020.0316 (2020).Gassart, A. d. et al. 442 ICT01, an anti-BTN3A mAb that activates Vg9Vd2 T cells, plus interleukin-2: a potent and promising combination for cancer immunotherapy. Journal for ImmunoTherapy of Cancer 8, A268-A269, doi:10.1136/jitc-2020-SITC2020.0442 (2020).Walkey, C., Swanson, R., Ulge, U., Silva Manzano, D. A. & Drachman, J. 576 NL-201, a de novo IL-2 and IL-15 agonist, demonstrates enhanced in vivo antitumor activity in combination with multiple cancer immunotherapies. Journal for ImmunoTherapy of Cancer 8, A346-A346, doi:10.1136/jitc-2020-SITC2020.0576 (2020).Walkey, C. D. et al. Abstract 4518: Pre-clinical development of NL-201: A de novo α-independent IL-2/IL-15 agonist. Cancer Research 80, 4518–4518, doi:10.1158/1538-7445.Am2020-4518 (2020).Ethics ApprovalAll procedures involving animals described in this study have been reviewed and approved by the local ethic committee (CELEAG) and the French Ministry of Research.
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