Scientific Resources

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here,we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state,followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. View Publication

BACKGROUND & AIMS N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS Mettl3 knockout mice,CD34+ humanized mice,and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay,respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells,and eventually suppressed CRC in ApcMin/+Mettl3+/- mice,CD34+ humanized mice,and syngeneic mice models. Mechanistically,METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing,RNA sequencing,and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner,which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However,the effect was negligible on BHLHE41 depletion,CXCL1 protein or CXCR2 inhibitor SB265610 administration,inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently,depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly,targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC. View Publication

BACKGROUND Acute respiratory distress syndrome (ARDS) is a neutrophil-associated disease. Delayed neutrophil apoptosis and increased levels of neutrophil extracellular traps (NETs) have been described in ARDS. We aimed to investigate the relationship between these phenomena and their potential as inflammation drivers. We hypothesized that delayed neutrophil apoptosis might enhance NET formation in ARDS. METHOD Our research was carried out in three aspects: clinical research,animal experiments,and in vitro experiments. First,we compared the difference between neutrophil apoptosis and NET levels in healthy controls and patients with ARDS and analyzed the correlation between neutrophil apoptosis and NET levels in ARDS. Then,we conducted animal experiments to verify the effect of neutrophil apoptosis on NET formation in Lipopolysaccharide-induced acute lung injury (LPS-ALI) mice. Furthermore,this study explored the relationship between neutrophil apoptosis and NETs at the cellular level. Apoptosis was assessed using morphological analysis,flow cytometry,and western blotting. NET formation was determined using immunofluorescence,PicoGreen assay,SYTOX Green staining,and western blotting. RESULTS ARDS neutrophils lived longer because of delayed apoptosis,and the cyclin-dependent kinase inhibitor,AT7519,reversed this phenomenon both in ARDS neutrophils and neutrophils in bronchoalveolar lavage fluid (BALF) of LPS-ALI mice. Neutrophils in a medium containing pro-survival factors (LPS or GM-CSF) form more NETs,which can also be reversed by AT7519. Tissue damage can be reduced by promoting neutrophil apoptosis. CONCLUSIONS Neutrophils with extended lifespan in ARDS usually enhance NET formation,which aggravates inflammation. Enhancing neutrophil apoptosis in ARDS can reduce the formation of NETs,inhibit inflammation,and consequently alleviate ARDS. View Publication

OBJECTIVE To identify immune cells,cytokines,and immune cell transcriptome in the menstrual effluent (ME) of women with endometriosis compared with that of healthy donors. DESIGN Live immune cells were isolated from human ME samples and were analyzed by flow cytometry to identify various immune cell populations. Selected cytokines from the same patients were evaluated using multiplex cytokine analyses. The transcriptome of the immune cell population was subsequently profiled using NanoString nCounter's PanCancer Immune panel. SETTING Academic institution. PATIENT(S) Surgically confirmed endometriosis patients (n = 14) and healthy fertile donors (n = 19). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) In-depth immune cell profiling of ME obtained from women with endometriosis compared with that of healthy donors. RESULT(S) ME analysis revealed that the number of T helper 17 (TH17) cells was significantly lower in patients with endometriosis compared with that of healthy donors; the number of macrophages was also lower (P=.06) in the former. Multiplex cytokine analysis revealed significantly lower transforming growth factor $\alpha$ in the ME serum" of patients with endometriosis. Transcriptomic analysis of CD45+ cells revealed 47 differentially expressed genes mainly associated with the TH17 axis (IL10 IL23A and IL6) as well as genes associated with macrophage signaling/activation (CD74 CD83 CXCL16 and CCL3). CONCLUSION(S) We demonstrate for the first time that the levels of TH17 axis macrophages and transforming growth factor $\alpha$ were altered in the ME of women with endometriosis compared with that of healthy donors. These findings shed light on the potential immune pathways that could partly explain the pathogenesis and progression of endometriosis. Future large-scale studies on ME samples are warranted to exploit the use of these markers to study the pathogenesis of endometriosis." View Publication

Gamma-delta (??) T cells recognize antigens in a major histocompatibility complex (MHC) independent and have cytotoxic capability. Human immunodeficiency virus (HIV) infection reduces the proportion of the V?2 cell subset compared to the V?1 cell subset of ?? T cells in the blood in most infected individuals,except for elite controllers. The capacity of V?2 T cells to kill HIV-infected targets has been demonstrated in vitro,albeit in vivo confirmatory studies are lacking. Here,we provide the first characterization of ?? T cell-HIV interactions in bone marrow-liver-thymus (BLT) humanized mice and examined the immunotherapeutic potential of V?2 T cells in controlling HIV replication in vivo. We demonstrate a reduced proportion of V?2 T cells and an increased proportion of V?1 T cells in HIV-infected BLT humanized mice,like in HIV-positive individuals. HIV infection in BLT humanized mice also impaired the ex vivo expansion of V?2 T cells,like in HIV-positive individuals. Adoptive transfer of activated V?2 T cells did not control HIV replication during cell-associated HIV transmission in BLT humanized mice but instead exacerbated viremia,suggesting that V?2 T cells may serve as early targets for HIV replication. Our findings demonstrate that BLT humanized mice can model ?? T cell-HIV interactions in vivo. View Publication

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however,a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study,we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically,GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells,GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity,resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis. View Publication

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease,the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study,a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus,preventing it from infecting cells in an in vitro cell-based assay,including multiple viral variants that are currently circulating in the human population. In addition,we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease. View Publication

Cytokine storm refers to the dysregulated production of inflammatory mediators leading to hyperinflammation. They are often detrimental,and worsen the severity of COVID-19 and other infectious or inflammatory diseases. Cannabinoids are known to have anti-inflammatory effects but their possible therapeutic value on cytokine storms has not been fully elucidated. In vivo and ex vivo studies were carried out to investigate the effects of high-THC and high-CBD extracts on cytokine production in immune cells. Significant differences between the extracts were observed. Subsequent experiments focusing on a specific high CBD extract (CBD-X) showed significant reductions in pro-inflammatory cytokines in human-derived PBMCs,neutrophils and T cells. In vivo mouse studies,using a systemically inflamed mouse model,showed reductions in pro-inflammatory cytokines TNF$\alpha$ and IL-1$\beta$ and a concurrent increase in the anti-inflammatory cytokine IL-10 in response to CBD-X extract treatment. Lung inflammation,as in severe COVID-19 disease,is characterized by increased T-cell homing to the lungs. Our investigation revealed that CBD-X extract impaired T-cell migration induced by the chemoattractant SDF1. In addition,the phosphorylation levels of T cell receptor (TCR) signaling proteins Lck and Zap70 were significantly reduced,demonstrating an inhibitory effect on the early events downstream to TCR activation. In a lung inflamed mouse model,we observed a reduction in leukocytes including neutrophil migration to the lungs and decreased levels of IL-1$\beta$,MCP-1,IL-6 and TNF$\alpha$,in response to the administration of the high-CBD extract. The results presented in this work offer that certain high-CBD extract has a high potential in the management of pathological conditions,in which the secretion of cytokines is dysregulated,as it is in severe COVID-19 disease or other infectious or inflammatory diseases. View Publication

We investigated genomic and transcriptomic changes in paired tumor samples of 29 in-house multiple myeloma (MM) patients and 28 patients from the MMRF CoMMpass study before and after treatment. A change in clonal composition was found in 46/57 (82%) of patients,and single-nucleotide variants (SNVs) increased from median 67 to 86. The highest increase in prevalence of genetic aberrations was found in RAS genes (60% to 72%),amp1q21 (18% to 35%),and TP53 (9% to 18%). The SBS-MM1 mutation signature was detected both in patients receiving high and low dose melphalan. A total of 2589 genes were differentially expressed between early and late samples (FDR?? View Publication

Conditioning regimens used for hematopoietic stem cell transplantation (HCT) can escalate the severity of acute T cell-mediated graft-versus-host disease (GVHD) by disrupting gastrointestinal integrity and initiating lipopolysaccharide (LPS)-dependent innate immune cell activation. Activation of the complement cascade has been associated with murine GVHD,and previous work has shown that alternative pathway complement activation can amplify T cell immunity. Whether and how mannan-binding lectin (MBL),a component of the complement system that binds mannose as well as oligosaccharide components of LPS and lipoteichoic acid,affects GVHD is unknown. In this study,we tested the hypothesis that MBL modulates murine GVHD and examined the mechanisms by which it does so. We adoptively transferred C3.SW bone marrow (BM) cells ± T cells into irradiated wild type (WT) or MBL-deficient C57Bl/6 (B6) recipients with or without inhibiting MBL-initiated complement activation using C1-esterase inhibitor (C1-INH). We analyzed the clinical severity of disease expression and analyzed intestinal gene and cell infiltration. In vitro studies assessed MBL expression on antigen-presenting cells (APCs) and compared LPS-induced responses of WT and MBL-deficient APCs. MBL-deficient recipients of donor BM ± T cells exhibited significantly less weight loss over the first 2 weeks post-transplantation weeks compared with B6 controls (P < .05),with similar donor engraftment in the 2 groups. In recipients of C3.SW BM + T cells,the clinical expression of GVHD was less severe (P < .05) and overall survival was better (P < .05) in MBL-deficient mice compared with WT mice. On day-7 post-transplantation,analyses showed that the MBL-deficient recipients exhibited less intestinal IL1b,IL17,and IL12 p40 gene expression (P < .05 for each) and fewer infiltrating intestinal CD11c+,CD11b+,and F4/80+ cells and TCR$\beta$+,CD4+,CD4+IL17+,and CD8+ T cells (P < .05 for each). Ovalbumin or allogeneic cell immunizations induced equivalent T cell responses in MBL-deficient and WT mice,demonstrating that MBL-deficiency does not directly impact T cell immunity in the absence of irradiation conditioning. Administration of C1-INH did not alter the clinical expression of GVHD in preconditioned WT B6 recipients,suggesting that MBL amplifies clinical expression of GVHD via a complement-independent mechanism. WT,but not MBL-deficient,APCs express MBL on their surfaces. LPS-stimulated APCs from MBL-deficient mice produced less proinflammatory cytokines (P < .05) and induced weaker alloreactive T cell responses (P < .05) compared with WT APCs. Together,our data show that MBL modulates murine GVHD,likely by amplifying complement-independent,LPS-initiated gastrointestinal inflammation. The results suggest that devising strategies to block LPS/MBL ligation on APCs has the potential to reduce the clinical expression of GVHD. View Publication

Ankylosing spondylitis (AS) is an immune-mediated inflammatory disorder that primarily affects the axial skeleton,especially the sacroiliac joints and spine. This results in chronic back pain and,in extreme cases,ankylosis of the spine. Despite its debilitating effects,the pathogenesis of AS remains to be further elucidated. This study used single cell CITE-seq technology to analyze peripheral blood mononuclear cells (PBMCs) in AS and in healthy controls. We identified a number of molecular features associated with AS. CD52 was found to be overexpressed in both RNA and surface protein expression across several cell types in patients with AS. CD16+ monocytes overexpressed TNFSF10 and IL-18R$\alpha$ in AS,while CD8+ TEM cells and natural killer cells overexpressed genes linked with cytotoxicity,including GZMH,GZMB,and NKG7. Tregs underexpressed CD39 in AS,suggesting reduced functionality. We identified an overrepresented NK cell subset in AS that overexpressed CD16,CD161,and CD38,as well as cytotoxic genes and pathways. Finally,we developed machine learning models derived from CITE-seq data for the classification of AS and achieved an Area Under the Receiver Operating Characteristic (AUROC) curve of > 0.95. In summary,CITE-seq identification of AS-associated genes and surface proteins in specific cell subsets informs our understanding of pathogenesis and potential new therapeutic targets,while providing new approaches for diagnosis via machine learning. View Publication

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