技术资料

MALT1 forms part of a central signaling node downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors,across a broad range of immune cell subsets,and regulates NF-$\kappa$B driven transcriptional responses via dual scaffolding-protease activity. Allosteric inhibition of MALT1 activity has demonstrated benefit in animal models of inflammation. However,development of MALT1 inhibitors to treat autoimmune and inflammatory diseases (A&ID) has been hindered by reports linking MALT1 inhibition and genetic loss-of-function to reductions in regulatory T-cell (Treg) numbers and development of auto-inflammatory syndromes. Using an allosteric MALT1 inhibitor,we investigated the consequence of pharmacological inhibition of MALT1 on proinflammatory cells compared to regulatory T-cells. Consistent with its known role in ITAM-driven responses,MALT1 inhibition suppressed proinflammatory cytokine production from activated human T-cells and monocyte-derived macrophages,and attenuated B-cell proliferation. Oral administration of a MALT1 inhibitor reduced disease severity and synovial cytokine production in a rat collagen-induced arthritis model. Interestingly,reduction in splenic Treg numbers was less pronounced in the context of inflammation compared with na{\{i}}ve animals. Additionally in the context of the disease model we observed an uncoupling of anti-inflammatory effects of MALT1 inhibition from Treg reduction with lower systemic concentrations of inhibitor needed to reduce disease severity compared to that required to reduce Treg numbers. MALT1 inhibition did not affect suppressive function of human Tregs in vitro. These data indicate that anti-inflammatory efficacy can be achieved with MALT1 inhibition without impacting the number or function of Tregs further supporting the potential of MALT1 inhibition in the treatment of autoimmune disease." View Publication

Most cancer vaccines target peptide antigens,necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore,tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells,but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding,enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably,this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary,highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations. View Publication

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses,and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1,attenuate differentiation,and enhance proliferation and antitumor efficacy in vivo. Here,we used a cell-free culture system to provide an agonistic NOTCH1 signal during na{\{i}}ve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation transcription factor expression cytokine production and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from na{\"{i}}ve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF driving heightened production of interleukin-22 interleukin-10 and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors." View Publication

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250??$\mu$g/ip day 0) and CTLA4-Ig (200??$\mu$g/ip day 2),we administered low-dose IL-2 (2000??IU/day) starting on posttransplant day 14 for 3??weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin,vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens,leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100??days),prevented chronic rejection,and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142,associated with the TGF$\beta$ pathway,was significantly downregulated in exosomes from IL-2-treated mice. In conclusion,low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules. View Publication

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here,we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation,oxidative phosphorylation,and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs,peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol,please refer to Mohammadpour et al. (2021). View Publication

OBJECTIVE The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa),this phenotype is reprogrammed,making it an interesting therapeutic target. However,how the truncated prostate TCA cycle works is still not completely understood. METHODS We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS First,mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate,thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis,several key nutrients were shown to allow the completion of the prostate TCA cycle,revealing a much more complex metabolic profile than originally anticipated. Indeed,along with the known pathway of aspartate replenishing oxaloacetate,glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids,aspartate entered the TCA cycle at the malate entry point,upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway,but also by at least three additional pathways: glutaminolysis,reductive carboxylation,and aspartate-malate conversion. CONCLUSIONS Our results add a significant new dimension to the prostate citrate-secretory phenotype,with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa. View Publication

BACKGROUND The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here,we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS To further understand CISH functions in NK cells,we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression,signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line,metastasis evaluation was performed. Then,orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally,we targeted CISH in human NK-92 or primary NK cells,using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS In Cishfl/flNcr1Ki/+ mice,we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways,triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor,optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression,limiting NK cell exhaustion. Finally,using CRISPRi,we then targeted CISH in human NK-92 or primary NK cells. In human NK cells,CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy. View Publication

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness,a process termed licensing or education,but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept,we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog,TMIGD2) and 2B4 (CD244,SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody,the CD28H transmembrane domain,and the fusion of CD28H,2B4,and TCR$\zeta$ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCR$\zeta$ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore,such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore,engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies,such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing. View Publication

Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here,we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs,while it was absent from CAFs. Furthermore,MAFs secreted higher levels of IGFBP2,CXCL2,CXCL6,CXCL12,PDGF-AA,FGFb,and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together,these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism. View Publication

BACKGROUND Hypoxia and inflammation tumor microenvironment (TME) play a crucial role in tumor development and progression. Although increased understanding of TME contributed to gastric cancer (GC) progression and prognosis,the direct interaction between macrophage and GC cells was not fully understood. METHODS Hypoxia and normoxia macrophage microarrays of GEO database was analyzed. The peripheral blood mononuclear cell acquired from the healthy volunteers. The expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) in GC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR),western-blot,Elisa and immunofluorescence. Cell proliferation,migration,and invasion were evaluated by cell counting kit 8 (CCK8),colony formation,real-time imaging of cell migration and transwell. Flow Cytometers was applied to identify the source of cytokines. Luciferase reporter assays and chromatin immunoprecipitation were used to identify the interaction between transcription factor and target gene. Especially,a series of truncated and mutation reporter genes were applied to identify precise binding sites. The corresponding functions were verified in the complementation test and in vivo animal experiment. RESULTS Our results revealed that hypoxia triggered macrophage secreted CXCL8,which induced GC invasion and proliferation. This macrophage-induced GC progression was CXCL8 activated C-X-C Motif Chemokine Receptor 1/2 (CXCR1/2) on the GC cell membrane subsequently hyperactivated Janus kinase 1/ Signal transducer and activator of transcription 1 (JAK/STAT1) signaling pathway. Then,the transcription factor STAT1 directly led to the overexpression and secretion of Interleukin 10 (IL-10). Correspondingly,IL-10 induced the M2-type polarization of macrophages and continued to increase the expression and secretion of CXCL8. It suggested a positive feedback loop between macrophage and GC. In clinical GC samples,increased CXCL8 predicted a patient's pessimistic outcome. CONCLUSION Our work identified a positive feedback loop governing cancer cells and macrophage in GC that contributed to tumor progression and patient outcome. View Publication

BACKGROUND miR-155-5p is associated with autoimmune diseases. T helper 17 (Th17) cells,interleukin (IL)-17,and suppressor of cytokines signaling 1 (SOCS1) have important roles in the pathogenesis of systemic sclerosis (SSc). The purpose of this study was to explore the role of miR-155-5p in the regulation of IL-17 and SOCS1 expression in Th17 cells and the subsequent effect on SSc disease progression. METHODS Th17 cells were isolated from peripheral blood mononuclear cells of SSc patients and healthy controls (HCs). RT-qPCR and western blotting were used to examine the expression patterns of miR-155-5p,IL-17,and SOCS1. Luciferase reporter assays were performed to confirm SOCS1 as a target of miR-155-5p. RNA pull-down assays were performed to detect the interaction of IL-17 and SOCS1 with miR-155-5p. In situ hybridization was performed to analyze the co-expression pattern of miR-155-5p and IL17A in Th17 cells. RESULTS The levels of Th17 cell-derived miR-155-5p were significantly up-regulated in SSc patients compared with HCs,and its levels were negatively correlated with SOCS1 levels. Meanwhile,miR-155-5p positively regulated IL-17 expression levels in Th17 cells isolated from SSc patients as the disease progressed. Using pmirGLO vectors,SOCS1 was confirmed as a target of miR-155-5p. The binding status of IL-17 and SOCS1 to miR-155-5p was related to SSc progression. An increase in the co-localization of miR-155-5p and IL-17 was associated with greater SSc progression. CONCLUSIONS IL-17 and SOCS1 expression modulated by Th17 cell-derived miR-155-5p are critical for SSc progression,which may provide novel insights into the pathogenesis of SSc. View Publication

BACKGROUND CD47/SIRP$\alpha$ axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer,particularly in hematological malignancies. In preclinical models,CD47/SIRP$\alpha$ blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here,we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry,transcriptomic analysis,and in vivo immune-cell depletion experiments. RESULTS We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells,monocytes,dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably,molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation,including IFN$\gamma$ and IL-12b. Moreover,TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701,highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition,NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro,resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS The study described the mechanisms afforded by the CD47xCD19 bispecific antibody,NI-1701,at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035). View Publication