Scientific Resources

Exosomes derived from mesenchymal stem cells (MSCs) could protect against myocardial infarction (MI). TLR4 is reported to play an important role in MI,while microRNA-182-5p (miR-182-5p) negatively regulates TLR4 expression. Therefore,we hypothesize that MSCs-derived exosomes overexpressing miR-182-5p may have beneficial effects on MI. We generated bone marrow mesenchymal stem cells (BM-MSCs) and overexpressed miR-182-5p in these cells for exosome isolation. H2O2-stimulated neonatal mouse ventricle myocytes (NMVMs) and MI mouse model were employed,which were subjected to exosome treatment. The expression of inflammatory factors,heart function,and TLR4 signaling pathway activation were monitored. It was found that miR-182-5p decreased TLR4 expression in BM-MSCs and NMVMs. Administration of exosomes overexpressing miR-182-5p to H2O2-stimulated NMVMs enhanced cell viability and suppressed the expression of inflammatory cytokines. In addition,they promoted heart function,suppressed inflammatory responses,and de-activated TLR4/NF-$\kappa$B signaling pathway in MI mice. In conclusion,miR-182-5p transferred by the exosomes derived from BM-MSCs protected against MI-induced impairments by targeting TLR4. View Publication

BACKGROUND Specific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA). METHODS RNA expression was assessed by NanoString in tumor samples of 41 treatment-na{\{i}}ve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion clonality and cytotoxic activity of expanded T cells. RESULTS 68.3% of patients expressed ??5 TAAs simultaneously with coregulated clusters which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ??1??TAA were detectable in 75.0% and 53.7% of patients respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare. CONCLUSIONS We identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA." 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 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

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

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

OBJECTIVES We sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens. METHODS Dendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24-72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs,cryopreserved T cells,and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing,2) expansion,and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs,we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4. RESULTS We observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However,polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore,resting these expanded T cells in IL-2,IL-7 or IL-15 did not restore the antigen dependent responses. In contrast,T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing. SIGNIFICANCE While screening techniques for potential pathological autoantibodies have come a long way,comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune 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