技术资料

OBJECTIVE True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. METHODS This was achieved by undertaking three sets of experiments. Firstly,SSEA-1+ and Oct-4??+??positive VSELs,sorted from GFP mice,were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly,uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24-72 h. Thirdly,OCT-4????expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5-7 after birth). RESULTS GFP??+??ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the estrus stage when maximum remodeling occurs. Mechanical scratching activated tissue-resident,nuclear OCT-4 positive VSELs and slightly bigger 'progenitors' endometrial stem cells (EnSCs,cytoplasmic OCT-4) which underwent clonal expansion and further differentiated into luminal and glandular epithelial cells. Neonatal exposure to endocrine disruption resulted in increased numbers of OCT-4 positive VSELs/EnSCs in adult endometrium. DISCUSSION Results support the presence of functionally active VSELs in adult endometrium. VSELs self-renew and give rise to EnSCs that further differentiate into epithelial cells under normal physiological conditions. Also,VSELs are vulnerable to endocrine insults. To conclude VSELs are true and elusive uterine stem cells that maintain life-long uterine homeostasis and their dysregulation may result in various pathologies. View Publication

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity,immunodeficiencies,and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies,which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-$\gamma$ (IFN-$\gamma$) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization,revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits,revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions,which could inform the design of immunotherapies. View Publication

Intronic single-nucleotide polymorphisms (SNPs) in the ANKRD55 gene are associated with the risk for multiple sclerosis (MS) and rheumatoid arthritis by genome-wide association studies (GWAS). The risk alleles have been linked to higher expression levels of ANKRD55 and the neighboring IL6ST (gp130) gene in CD4+ T lymphocytes of healthy controls. The biological function of ANKRD55,its role in the immune system,and cellular sources of expression other than lymphocytes remain uncharacterized. Here,we show that monocytes gain capacity to express ANKRD55 during differentiation in immature monocyte-derived dendritic cells (moDCs) in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). ANKRD55 expression levels are further enhanced by retinoic acid agonist AM580 but downregulated following maturation with interferon (IFN)-$\gamma$ and lipopolysaccharide (LPS). ANKRD55 was detected in the nucleus of moDC in nuclear speckles. We also analyzed the adjacent IL6ST,IL31RA,and SLC38A9 genes. Of note,in healthy controls,MS risk SNP genotype influenced ANKRD55 and IL6ST expression in immature moDC in opposite directions to that in CD4+ T cells. This effect was stronger for a partially correlated SNP,rs13186299,that is located,similar to the main MS risk SNPs,in an ANKRD55 intron. Upon analysis in MS patients,the main GWAS MS risk SNP rs7731626 was associated with ANKRD55 expression levels in CD4+ T cells. MoDC-specific ANKRD55 and IL6ST mRNA levels showed significant differences according to the clinical form of the disease,but,in contrast to healthy controls,were not influenced by genotype. We also measured serum sgp130 levels,which were found to be higher in homozygotes of the protective allele of rs7731626. Our study characterizes ANKRD55 expression in moDC and indicates monocyte-to-dendritic cell (Mo-DC) differentiation as a process potentially influenced by MS risk SNPs. View Publication

Cx3cr1+ monocyte-derived macrophages (moMacs) are recruited to tissues after injury and are known to have profibrotic effects,but the cell-cell interactions and specific pathways that regulate this polarization and function are incompletely understood. Here we investigate the role of moMac-derived Pdgfa in bleomycin-induced lung fibrosis in mice. Deletion of Pdgfa with Cx3cr1-CreERT2 decreased bleomycin-induced lung fibrosis. Among a panel of in vitro macrophage polarizing stimuli,robust induction of Pdgfa was noted with IL10 in both mouse and human moMacs. Likewise,analysis of single-cell data revealed high expression of the receptor IL10RA in moMacs from human fibrotic lungs. Studies with IL10-GFP mice revealed that IL10-expressing cells were increased after injury in mice and colocalized with moMacs. Notably,deletion of IL10ra with Csf1r-Cre: IL10ra fl/fl mice decreased both Pdgfa expression in moMacs and lung fibrosis. Taken together,these findings reveal a novel,IL10-dependent mechanism of macrophage polarization leading to fibroblast activation after injury. View Publication

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures,where long single-stranded DNA is folded into a compact nanostructure,present an attractive approach to package genes; however,effective delivery of genetic material into cell nuclei has remained a critical challenge. Here,we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore,our study validates virus-like particles as an efficient method of DNA nanostructure delivery,opening the possibility of delivering nanostructures in vivo to specific cell types. Together,these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates,exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions,such as biosensing,into cell nuclei. View Publication

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus,restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis,in addition to standard clinical care. Herein,we evaluated the effects of the SPM lipids,lipoxin A4 (LXA4 ) and lipoxin B4 (LXB4 ),on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production,and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst,a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls,indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin,which plays a central role in clot activation and atherosclerosis. Finally,LXB4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy,as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function,LXB4 displayed more potent effects than LXA4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual. View Publication

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses,development of therapeutics that target Tregs is limited by their low abundance,heterogeneity,and lack of specific cell surface markers. We isolated human PBMC-derived CD4+ CD25high Foxp3+ Tregs and demonstrate they suppress stimulated CD4+ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining,we identified a human T cell line,MoT,as a model of human Foxp3+ Tregs. Unlike Jurkat T cells,MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4,CD25,GITR,LAG-3,PD-L1,CCR4. PBMC-derived Tregs and MoT cells,but not Jurkat cells,inhibited proliferation of human CD4+PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4+PBMC proliferation by MoT cells and Tregs,suggesting cell-cell contact is required for suppressive activity. Blocking antibodies against PD-L1,LAG-3,GITR,CCR4,HLA-DR,or CTLA-4 did not reverse the suppressive activity.We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4+PBMCs in a cell contact-dependent manner,suggesting that a Foxp3+Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism. View Publication

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses,therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here,we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs),squeezed DCs were ˆ¼1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs,such as T cells,for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells,such as murine splenocytes,also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs,T cells,B cells,and PBMCs and that,in clinical scale formats,the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model,TC-1,we demonstrate that squeezed B cells,T cells,and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together,these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients. View Publication

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses,which led to a recent US Food and Drug Administration approval. Despite the success of this therapy,durable remissions continue to be low,and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models,we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7,which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition,CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM. View Publication

Sialic acids are negatively charged carbohydrates that cap the glycans of glycoproteins and glycolipids. Sialic acids are involved in various biological processes including cell-cell adhesion and immune recognition. In dendritic cells (DCs),the major antigen-presenting cells of the immune system,sialic acids emerge as important regulators of maturation and interaction with other lymphocytes including T cells. Many aspects of how sialic acids regulate DC functions are not well understood and tools and model systems to address these are limited. Here,we have established cultures of murine bone marrow-derived DCs (BMDCs) that lack sialic acid expression using a sialic acid-blocking mimetic Ac53FaxNeu5Ac. Ac53FaxNeu5Ac treatment potentiated BMDC activation via toll-like receptor (TLR) stimulation without affecting differentiation and viability. Sialic acid blockade further increased the capacity of BMDCs to induce antigen-specific CD8+ T cell proliferation. Transcriptome-wide gene expression analysis revealed that sialic acid mimetic treatment of BMDCs induces differential expression of genes involved in T cell activation,cell-adhesion,and cell-cell interactions. Subsequent cell clustering assays and single cell avidity measurements demonstrated that BMDCs with reduced sialylation form higher avidity interactions with CD8+ T cells. This increased avidity was detectable in the absence of antigens,but was especially pronounced in antigen-dependent interactions. Together,our data show that sialic acid blockade in BMDCs ameliorates maturation and enhances both cognate T cell receptor-MHC-dependent and independent T cell interactions that allow for more robust CD8+ T cell responses. View Publication

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly,this type of immunoglobulin usually pairs with the single germline VL gene,V30 that is typically very conserved in sequence. In this work,we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection,EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly,engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-$\gamma$. Taken together,this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications. View Publication

Oncogenic activated RAS mutations have been detected in 50% of de novo and 70% of relapsed multiple myeloma (MM) patients. Translocation t(11;14) involving IgH/CCDN1 and overexpression of cyclin-Ds are early events in MM pathogenesis,enhancing uncontrolled MM cell growth. We hypothesized that targeting both RAS/MAPK pathway molecules including Erk1/2 along with cyclin-Ds enhances MM cytotoxicity and minimizes side effects. Recent studies have demonstrated the high potency of Erk1/2 and CDK4/6 inhibitors in metastatic relapsed cancers,and here we tested anti-MM effects of the Erk1/2??+??CDK4/6 inhibitor combination. Our studies showed strong synergistic (IC?? View Publication