技术资料

Neutrophils are central players in the innate immune system. To protect against invading pathogens,neutrophils can externalize chromatin to create neutrophil extracellular traps (NETs). While NETs are critical to host defense,they also have deleterious effects,and dysregulation of NETs formation has been implicated in autoimmune diseases,atherosclerosis and thrombotic conditions,cancer progression and dissemination,and acute respiratory distress syndrome. Here,we report that selinexor,a first-in-class selective inhibitor of nuclear export approved for the treatment of multiple myeloma and diffuse large B-cell lymphoma,markedly suppressed the release of NETs in vitro. Furthermore,we demonstrate a significant inhibitory effect of selinexor on NETs formation,but not on oxidative burst or enzymatic activities central to NETs release such as neutrophil elastase,myeloperoxidase or peptidyl arginine deiminase type IV. The inhibitory effect of selinexor was demonstrated in neutrophils activated by a variety of NETs-inducers,including PMA,TGF-$\beta$,TNF-$\alpha$ and IL-8. Maximal inhibition of NETs formation was observed using TGF-$\beta$,for which selinexor inhibited NETs release by 61.6%. These findings pave the way to the potential use of selinexor in an effort to reduce disease burden by inhibition of NETs. View Publication

BACKGROUND Suppression of cardiac iinflammasome,which can be inhibited by Farnesoid X receptor (FXR) agonist,can ameliorate cardiac inflammation and fibrosis. Increased cardiac inflammasome decrease the abundance of regulatory T (Treg) cells and exacerbate cardiac dysfunction. Interaction between cardiomyocytes and Treg cells is involved in the development of nonalcoholic steatohepatitis (NASH)-related cardiac dysfunction. AIMS This study evaluates whether the FXR agonist obeticholic acid (OCA) treatment improves NASH-associated cardiac dysfunction. METHODS The in vivo and in vitro mechanisms and effects of two weeks of OCA treatment on inflammasome and Treg dysregulation-related cardiac dysfunction in NASH mice (NASH-OCA) at systemic,tissue and cellular levels were investigated. RESULTS The OCA treatment suppressed the serum and cardiac inflammasome levels,reduced the cardiac infiltrated CD3+ T cells,increased the cardiac Treg-represented anti-inflammatory cytokines (IL-10/IL-10R) and improved cardiac inflammation,fibrosis and function [decreased left ventricle (LV) mass and increased fractional shortening (FS)] in NASH-OCA mice. The percentages of OCA-decreased cardiac fibrosis and OCA-increased FS were positively correlated with the percentage of OCA-increased levels of cardiac FXR and IL-10/IL-10R. In the Treg cells from NASH-OCA mice spleen,in comparison with the Treg cells of the NASH group,higher intracellular FXR but lower inflammasome levels,and more proliferative/active and less apoptotic cells were observed. Incubation of H9c2 cardiomyoblasts with Treg-NASHcm [supernatant of Treg from NASH mice as condition medium (cm)],increased inflammasome levels,decreased the proliferative/active cells,suppressed the intracellular FXR,and downregulated differentiation/contraction marker. The Treg-NASHcm-induced hypocontractility of H9c2 can be attenuated by co-incubation with OCA,and the OCA-related effects were abolished by siIL-10R pretreatment. CONCLUSIONS Chronic FXR activation with OCA is a potential strategy for activating IL-10/IL-10R signalling,reversing cardiac regulatory T cell dysfunction,and improving inflammasome-mediated NASH-related cardiac dysfunction. View Publication

INTRODUCTION Based on the immunologic effects of anti-cancer treatment and their therapeutic implications,we evaluated radiotherapy (RT)-induced dynamic alterations in programmed death-1 (PD-1)/PD ligand-1 (PD-L1) expression profiles. METHODS Local RT with 2 Gy ?— 5 or 7.5 Gy ?— 1 was administered to the CT26 mouse model. Thereafter,tumors were resected and evaluated at the following predefined timepoints according to radiation response status: baseline,early (immediately after RT),middle (beginning of tumor shrinkage),late (stable status with RT effect),and progression (tumor regrowth). PD-1/PD-L1 activity and related immune cell profiles were quantitatively assessed. RESULTS RT upregulated PD-L1 expression in tumor cells from the middle to late phase; however,the levels subsequently decreased to levels comparable to baseline in the progression phase. RT with 2 Gy ?— 5 induced a higher frequency of PD-L1+ myeloid-derived suppressor cells,with a lesser degree of tumor regression,compared to 7.5 Gy. The proportion of PD-1+ and interferon (IFN)-$\gamma$+CD8$\alpha$ T cells continued to increase. The frequency of splenic PD-1+CD8+ T cells was markedly elevated,and was sustained longer with 2 Gy ?— 5. Based on the transcriptomic data,RT stimulated the transcription of immune-related genes,leading to sequentially altered patterns. DISCUSSION The dynamic alterations in PD-1/PD-L1 expression level were observed according to the time phases of tumor regression. This study suggests the influence of tumor cell killing and radiation dosing strategy on the tumor immune microenvironment. View Publication

Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here,we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample,and 10?— Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein,we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development. For complete details on the use and execution of this protocol,please refer to Nozais et al. (2021). View Publication

Regulatory T cells (Tregs) are critically involved in neovascularization,an important compensatory mechanism in peripheral artery disease. The contribution of G protein coupled receptor 174 (GPR174),which is a regulator of Treg function and development,in neovascularization remains elusive. Here,we show that genetic deletion of GPR174 in Tregs potentiated blood flow recovery in mice after hindlimb ischemia. GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs,thereby enhancing endothelial cell functions and reducing pro-inflammatory macrophage polarization and endothelial cell apoptosis. Mechanically,GPR174 regulates AREG expression by inhibiting the nuclear accumulation of early growth response protein 1 (EGR1) via G$\alpha$s/cAMP/PKA signal pathway activation. Collectively,these findings demonstrate that GPR174 negatively regulates angiogenesis and vascular remodeling in response to ischemic injury and that GPR174 may be a potential molecular target for therapeutic interventions of ischemic vascular diseases. View Publication

Previous studies demonstrated that CD4+ T cells can uptake tumor antigen-pulsed dendritic cell-derived exosomes (DEXO),which harbor tumor antigen peptide/pMHC I complex and costimulatory molecules and show potent effects on inducing antitumor immunity. However,in preliminary study,CD4+ T cells targeted by leukemia cell-derived exosomes (LEXs) did not show the expected effects in inducing effective anti-leukemia immunity,indicating that LEX is poorly immunogenetic largely due to an inadequate costimulatory capacity. Therefore,LEX-based anti-leukemia vaccines need to be optimized. In this study,we constructed a novel LEX-based vaccine by combining CD4+ T cells with costimulatory molecules gene-modified LEXs,which harbor upregulated CD80 and CD86,and the anti-leukemia immunity of CD80 and CD86 gene-modified LEX-targeted CD4+ T cells was investigated. We used lentiviral vectors encoding CD80 and CD86 to successfully transduced the L1210 leukemia cells,and the expression of CD80 and CD86 was remarkably upregulated in leukemia cells. The LEXs highly expressing CD80 and CD86 were obtained from the supernatants of gene-transduced leukemia cells. Our data have shown that LEX-CD8086 could promote CD4+ T cell proliferation and Th1 cytokine secretion more efficiently than control LEXs. Moreover,CD4+ TLEX-CD8086 expressed the acquired exosomal costimulatory molecules. With acquired costimulatory molecules,CD4+ TLEX-CD8086 can act as APCs and are capable of directly stimulating the leukemia cell antigen-specific CD8+ CTL response. This response was higher in potency compared to that noted by the other formulations. Furthermore,the animal study revealed that the CD4+ TLEX-CD8086 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice than other formulations did in both protective and therapeutic models. In conclusion,this study revealed that CD4+ TLEX-CD8086 could effectively induce more potential anti-leukemia immunity than LEX-CD8086 alone,suggesting that the utilization of a costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine may have promising potential for leukemia immunotherapy. View Publication

Rationale: IgA can induce activation of neutrophils which are the most abundant cell type in blood,but the development of IgA as therapeutic has been confounded by its short half-life and a weak ability to recruit NK cells as effector cells. Therefore,we generated an X-shaped antibody (X-body) based on the principle of molecular self-assembly that combines the activities of both IgG and IgA,which can effectively recruit and activate NK cells,macrophages,and neutrophils to kill tumor cells. Methods: X-body was generated by using a self-assembly strategy. The affinity of the X-body with the antigen and Fc receptors was tested by surface plasmon resonance. The shape of X-body was examined using negative staining transmission electron microscopy. The tumor cell killing activity of X-body was assessed in vitro and in multiple syngeneic mouse models. To explore the mechanism of X-body,tumor-infiltrating immune cells were analyzed by single-cell RNA-seq and flow cytometry. The dependence of neutrophil,macrophage,and NK cells for the X-body efficacy was confirmed by in vivo depletion of immune cell subsets. Results: The X-body versions of rituximab and trastuzumab combined the full spectrum activity of IgG and IgA and recruited NK cells,macrophages,and neutrophils as effector cells for eradication of tumor cells. Treatment with anti-hCD20 and anti-hHER2 X-bodies leads to a greater reduction in tumor burden in tumor-bearing mice compared with the IgA or IgG counterpart,and no obvious adverse effect is observed upon X-body treatment. Moreover,the X-body has a serum half-life and drug stability comparable to IgG. Conclusions: The X-body,as a myeloid-cell-centered therapeutic strategy,holds promise for the development of more effective cancer-targeting therapies than the current state of the art. View Publication

Normal density granulocytes (NDGs) can suppress T-cell responses in a similar way as myeloid-derived suppressor cells (MDSCs). In this study,we tested the hypothesis that NDGs from healthy donors preferentially inhibit T helper 1 (Th1) cells and investigated the myeloid-derived suppressive effect in different T-cell populations. We found that NDG-induced suppression of T-cell proliferation was contact dependent,mediated by integrin CD11b,and dependent on NDG-production of reactive oxygen species (ROS). The suppression was rapid and occurred within the first few hours of coculture. The suppression did not influence the CD8+/CD4+ ratio indicating an equal sensitivity in these populations. We further analyzed the CD4+ T helper subsets and found that NDGs induced a loss of Th1 surface marker,CD183,that was unrelated to ligand-binding to CD183. In addition,we analyzed the Th1,Th2,and Th17 cytokine production and found that all cytokine groups were suppressed when T-cells were incubated with NDGs. We therefore concluded that NDGs do not preferentially suppress Th1-cells. Instead,NDGs generally suppress Th cells and cytotoxic T-cells but specifically downregulate the Th1 marker CD183. View Publication

Facile and sensitive detection and isolation of circulating tumor cells (CTCs) was achieved using the aptamer-targeted magnetic nanoparticles (Apt-MNPs) in conjugation with a microfluidic device. Apt-MNPs were developed by the covalent attachment of anti-MUC1 aptamer to the silica-coated magnetic nanoparticles via the glutaraldehyde linkers. Apt-MNPs displayed high stability and functionality after 6 months of storage at 4 °C. The specific microfluidic device consisting of mixing,sorting and separation modules was fabricated through conventional photo- and soft-lithography by using polydimethylsiloxane. The capture efficiency of Apt-MNPs was first studied in vitro on MCF-7 and MDA-MB-231 cancer cell lines in the bulk and microfluidic platforms. The cell capture yields of more than 91% were obtained at the optimum condition after 60 minutes of exposure to 50 $\mu$g mL-1 Apt-MNPs with 10 to 106 cancer cells in different media. CTCs were also isolated efficiently from the blood samples of breast cancer patients and successfully propagated in vitro. The isolated CTCs were further characterized using immunofluorescence staining. The overall results indicated the high potential of the present method for the detection and capture of CTCs. View Publication

A breakdown in cellular homeostasis is thought to drive na{\{i}}ve T cell aging however the link between na{\"{i}}ve T cell homeostasis and aging in humans is poorly understood. To better address this we developed a lymphoid organoid system that maintains resting na{\"{i}}ve T cells for more than 2 weeks in conjunction with high CD45RA expression. Deep phenotypic characterization of na{\"{i}}ve T cells across age identified reduced CD45RA density as a hallmark of aging. A conversion from CD45RAhigh naive cells to a CD45RAlow phenotype was reproduced within our organoid system by structural breakdown but not by stromal cell aging or reduced lymphocyte density and mediated by alternative CD45 splicing. Together these data suggest that external influences within the lymph node microenvironment may cause phenotypic conversion of na{\"{i}}ve T cells in older adults." View Publication

BACKGROUND CD8+ T cells are important for protective immunity against intracellular pathogens. Excessive amounts of antigen and/or inflammatory signals often lead to the gradual deterioration of CD8+ T cell function,a state called exhaustion". However the association between CD8+ T cell exhaustion and acute respiratory distress syndrome (ARDS) has not been studied. This study was conducted to elucidate how CD8+ T cells and inhibitory receptors were related to the clinical prognosis of ARDS. METHODS A prospective observational study in an emergency department enrolled patients who were diagnosed with sepsis-associated ARDS according to the sepsis-3 criteria and Berlin definition. Peripheral blood samples were collected within 24??h post recruitment. CD8+ T cell count proliferation ratio cytokine secretion and the expression of coinhibitory receptors were assayed. RESULTS Sixty-two patients with ARDS met the inclusion criteria. CD8+ T cell counts and proliferation rates were dramatically decreased in non-surviving ARDS patients. Increasing programmed cell death 1 (PD-1) expression on the CD8+ T cell surface was seen in patients with worse organ function while an increasing level of T cell immunoglobulin mucin-3 (Tim-3) was associated with a longer duration of the shock. Kaplan-Meier analysis showed that low CD8+ T cell percentages and increased inhibitory molecule expression were significantly associated with a worse survival rate. CONCLUSIONS CD8+ T cells and coinhibitory receptors are promising independent prognostic markers of sepsis-induced ARDS and increased CD8+ T cell exhaustion is significantly correlated with poor prognosis." View Publication

Same day processing of biospecimens such as blood is not always feasible,which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end,whole blood was collected from healthy control subjects and processed fresh or at 6,24 and 48 h after collection and handling under modeled shipping conditions. At endpoint,whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR,CD66b,CD3,CD14,CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 h at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 h,although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level,RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However,these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 h at ambient temperature. However,neutrophil responses were largely abrogated by this time. By 48 h neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 h of collection,yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research,this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms. View Publication