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Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here,we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs,which are the most potent subsets,it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol,please refer to Liang et al. (2020) and Qiu et al. (2021). View Publication

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless,detection of spurious reactivities remains a recognized assay limitation. In addition,the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique,which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed,with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly,negligible reactivity was detected in the last wash control,confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly,antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis,AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity. View Publication

BACKGROUND Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far,the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD,and compare them to blood neutrophils. METHODS Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n??=??32) and a control group of patients with non-PCD,non-cystic fibrosis bronchiectasis (n??=??5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization,production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production,actin polymerization and the expulsion of NETs. In addition,PCD sputum displayed high activity of neutrophil elastase,and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD. View Publication

BACKGROUND There is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However,there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further,it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages. RESULTS We used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell,CD4 T,and CD8 T na{\{i}}ve and memory cells states. In the process of na{\"{i}}ve to memory activation across the three lineages we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further in central to effector memory differentiation our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes. CONCLUSIONS Our data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes." View Publication

BACKGROUND Myeloid-derived suppressor cells (MDSCs) can potently inhibit T-cell activity,promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive activities is intimately associated with cancer immunotherapy. Natural killer (NK) cells can engage in crosstalk with multiple myeloid cells to alter adaptive immune responses,triggering T-cell immunity. However,whether the NK-cell-MDSC interaction can modulate the T-cell immune response requires further study. Cryo-thermal therapy could induce the maturation of MDSCs by creating an acute inflammatory environment to elicit a CD4+ Th1-dominant immune response,but the mechanism regulating this process remains unclear. METHODS NK cells were depleted and NKG2D was blocked with monoclonal antibodies in vivo. MDSCs,NK cells and T cells were assessed by flow cytometry and isolated by magnetic-activated cell sorting (MACS). MDSCs and NK cells were cocultured with T cells to determine their immunological function. The transcriptional profiles of MDSCs were measured by qRT-PCR and RNA-sequencing. Isolated NK cells and MDSCs by MACS were cocultured to study the viability and maturation of MDSCs regulated by NK cells. TIMER was used to comprehensively examine the immunological,clinical,and genomic features of tumors. RESULTS NK-cell activation after cryo-thermal therapy decreased MDSC accumulation and reprogrammed immunosuppressive MDSCs toward a mature phenotype to promote T cell antitumor immunity. Furthermore,we discovered that NK cells could kill MDSCs via the NKG2D-NKG2DL axis and promote MDSC maturation by interferon gamma (IFN-$\gamma$) in response to NKG2D. In addition,CD4+ Th1-dominant antitumor immune response was dependent on NKG2D,which promoted the major histocompatibility complex …¡ pathway of MDSCs. High activated NK-cell infiltration and NKG2D level in tumors were positively correlated with better clinical outcomes. CONCLUSIONS Cryo-thermal therapy induces effective CD4+ Th1-dominant antitumor immunity by activating NK cells to reprogram MDSCs,providing a promising therapeutic strategy for cancer immunotherapy. View Publication

Asthmatic women tend to develop severe airway disease in their reproductive years,and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells,or ILC2,are readily detected in allergic conditions,such as rhinosinusitis,in individuals that develop nasal polyps do to allergen exposures,and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33,an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in na{\{i}}ve and ovalbumin (OVA) challenged female BALB/c mice in comparison to male mice following stimulation with IL-33. Here we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-$\kappa$B signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated and had reduced NF-$\kappa$B activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2 and Nfkb1 was found among top-ranked regulatory interactions. Together these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases." View Publication

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

Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF) and their precursor mesenchymal stromal cells (MSC) are often detected together in tumors,but how they cooperate is not well understood. Here,we show that TAM and CAF are the most abundant nonmalignant cells and are present together in untreated human neuroblastoma (NB) tumors that are also poorly infiltrated with T and natural killer (NK) cells. We then show that MSC and CAF-MSC harvested from NB tumors protected human monocytes (MN) from spontaneous apoptosis in an interleukin (IL)-6 dependent mechanism. The interactions of MN and MSC with NB cells resulted in a significant induction or increase in the expression of several pro-tumorigenic cytokines/chemokines (TGF-$\beta$1,MCP-1,IL-6,IL-8,and IL-4) but not of anti-tumorigenic cytokines (TNF-$\alpha$,IL-12) by MN or MSC,while also inducing cytokine expression in quiescent NB cells. We then identified a TGF-$\beta$1/IL-6 pathway where TGF-$\beta$1 stimulated the expression of IL-6 in NB cells and MSC,promoting TAM survival. Evidence for the contribution of TAM and MSC to the activation of this pathway was then provided in xenotransplanted NB tumors and patients with primary tumors by demonstrating a direct correlation between the presence of CAF and p-SMAD2 and p-STAT3. The data highlight a new mechanism of interaction between TAM and CAF supporting their pro-tumorigenic function in cancer. 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