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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

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

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

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

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The anti-diabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention,although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1),a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of anti-apoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels,which favors cytotoxic lymphocytes binding to tumor cells. Finally,metformin decreases the growth of human hematological tumor cells in xenograft models,mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy. View Publication

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study,we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively,while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs,multiple uniquely shared amino acid configurations were identified,warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes,which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation. View Publication

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL),but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission,we developed a dual-targeting approach using an optimized,bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release,degranulation,and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants,whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors,respectively. Further,CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence,raising the possibility that these cells may have the potential to promote durable remissions. Together,our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL. View Publication

BACKGROUND Adoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors,but its clinical application has been stalled because of toxicity. Here,we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors. METHODS We generated a cell membrane-anchored IL-12 (aIL12),a tumor-targeted IL-12 (ttIL12),and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells,chimeric antigen receptor-T cells,and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells. RESULTS attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically,attIL12-T cells targeted tumor cells expressing cell-surface vimentin,enriching effector T cell and interferon $\gamma$ production in tumors,which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects. CONCLUSIONS This novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors. View Publication

Early erythroid progenitors are transit-amplifying cells with high proliferative capacity committed to undergoing red cell differentiation. CD71/CD24low progenitors are less mature and have greater proliferative capacity than CD71/CD24high. We present protocols for isolation of CD71/CD24low progenitors from mouse fetal liver using both fluorescence-activated cell sorting (FACS) and immunomagnetic enrichment. CD71/CD24low progenitors isolated with both approaches show similar transcriptomes at single-cell resolution and exhibit characteristic proliferative responses to glucocorticoids. For complete details on the use and execution of this protocol,please refer to Li et al. (2019). View Publication