Surface Marker Expression Research Articles

Flow Cytometry of CD5-Positive Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder for which diagnosis is typically straightforward, based on bone marrow morphology and flow cytometry (FC) or immunohistochemistry. Nevertheless, variants present atypical expressions of cell surface markers, as is the case of CD5, for which the differential diagnosis can be more difficult. The aim of the current paper was to describe diagnosis of HCL with atypical CD5 expression, with an emphasis on FC. The detailed diagnostic methodology for HCL with atypical CD5 expression is presented, including differential diagnosis from other lymphoproliferative diseases with similar pathologic features, by FC analysis of the bone marrow aspirate. Diagnosis of HCL by means of FC started by gating all events based on side scatter (SSC) versus CD45 and B lymphocytes were selected from the lymphocytes gate as CD45/CD19 positive. The gated cells were positive for CD25, CD11c, CD20, and CD103, while CD10 proved to be dim to negative. Moreover, cells positive for CD3, CD4, and CD8, the three pan-T markers, as well as CD19, showed a bright expression of CD5. The atypical CD5 expression is usually correlated with a negative prognosis and thus chemotherapy with cladribine should be initiated. HCL is an indolent chronic lymphoproliferative disorder and diagnosis is usually straightforward. However, atypical expression of CD5 renders its differential diagnosis more difficult, but FC is a useful tool that allows an optimal classification of the disease and allows initiation of timely satisfactory therapy.

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Ultrasound cavitation: a reliable non-enzymatic method for adipose-derived mesenchymal stem cell (ADSC) isolation

BackgroundAdipose tissue is known to serve as an abundant and readily accessible source of adipose-derived stem cells (ADSCs) as an alternative to bone marrow. Collagenase is one of the most widely used methods for the isolation of ADSCs from adipose tissue, but it takes a long time, and there are also debates about safety. We propose an ultrasonic cavitation-treated method that can significantly reduce time and avoid the problem of using xenogeneic enzymes in ADSCs isolation.MethodsADSCs were isolated from adipose tissue using the enzyme treatment method and the ultrasonic cavitation treatment method. Cell proliferation was measured using cell viability assay. The expression levels of the surface markers of ADSCs were estimated by real-time PCR. After, ADSCs were cultured in chondrogenic, osteogenic, or adipogenic differentiation medium; the differentiation potential of ADCSs was analyzed by Alcian blue, Alizarin Red S, Oil Red O, and real-time PCR.ResultsThe cells treated with collagenase and ultrasound had similar cell yields and proliferation after isolation. The difference in the expression of surface markers of ADSCs was not statistically significant. ADSCs showed differentiation potential into adipocytes, osteocytes, and chondrocytes, and there was no difference between the enzyme treatment method and the ultrasonic cavitation treatment method. The yield of the ADSC increased in time- and intensity dependently.ConclusionsUltrasound certainly serves as a promising method in advancing ADSC isolation technology.

Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells

BackgroundWe evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient.MethodsEPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban.ResultsRivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair.ConclusionsRivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

Platelet-Derived Growth Factor-BB Priming Enhances Vasculogenic Capacity of Bone Marrow-Derived Endothelial Precursor Like Cells.

Human endothelial progenitor cells (EPCs) were first identified in the peripheral blood and later in the cord blood and bone marrow (BM) with different vascularization capacity and different surface marker profiles. However, their identity and functional roles in neovascularization have not been clearly demonstrated in vivo and in vitro. Characterization of BM-EPC like cells were performed by fluorescence-activated cell sorting, immunofluorescence staining, enzyme-linked immunosorbent assay, Matrigel tube formation assay,andwestern blot analysis. BM-EPC like cells were identified by selective adhesion to fibronectin and collagen from BM mononuclear cells, which generate fast-growing colonies with spindle morphology, express surface markers of CD105, vWF, UEA-I lectin binding, secrete HGF, VEGF, TGF-beta1 but can be distinguished from circulating EPC and endothelial cells by no expression of surface markers such as CD31, CD309, CD45, and CD34. These BM-EPC like cells shared many cell surface markers of BM-mesenchymal stem cells (MSC) but also can be distinguished by their vasculogenic property and other unique surface markers. Furthermore, the vasculogenic capacity of BM-EPC like cells were enhanced by co-culture of BM-MSC or PDGF-BB priming. PDGF-BB stimulated cell migration, proliferation, and secretion of laminin β-1, which was proposed as one of the mechanisms involved in the better vascularization of BM-EPC like cells. PDGF-BB priming may be applied to improve the potency and function of BM-EPC like cells as vasculogenic cell therapy for the ischemic vascular repair.

Application of human iPSC-derived macrophages in a miniaturized high-content-imaging-based efferocytosis assay.

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages. These IDM cells resembled MDMs both with respect to surface marker expression and phago- as well as efferocytotic function. A statistically robust high-content-imaging assay was developed to quantify the efferocytosis rate of IDMs and MDMs allowing for measurements both in the 384- and 1536-well microplate format. Validating the applicability of the assay, inhibitors of spleen tyrosine kinase (Syk) were shown to modulate efferocytosis in IDMs and MDMs with comparable pharmacology. The miniaturized cellular assay with the upscaled provision of macrophages opens new routes to pharmaceutical drug discovery in the context of efferocytosis-modulating substances.

Comparison of Biological Characteristics of Human Umbilical Cord Wharton’s Jelly-Derived Mesenchymal Stem Cells from Extremely Preterm and Term Infants

Background:Despite the progress in perinatal-neonatal medicine, complications of extremely preterm infants continue to constitute the major adverse outcomes in neonatal intensive care unit. Human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells (HUMSCs) may offer new hope for the treatment of intractable neonatal disorders. This study will explore the functional differences of HUMSCs between extremely preterm and term infants.Methods:UMSCs from 5 extremely preterm infants(weeks of gestation: 22+5 w,24+4 w,25+3 w,26 w,28 w) and 2 term infants(39 w,39+2 w) were isolated, and mesenchymal markers, pluripotent genes, proliferation rate were analyzed. HUVECs were injured by treated with LPS and repaired by co-cultured with HUMSCs of different gestational ages.Results:All HUMSCs showed fibroblast-like adherence to plastic and positively expressed surface marker of CD105,CD73 and CD90, but did not expressed CD45,CD34,CD14,CD79a and HLA-DR; HUMSCs in extremely preterm exhibited significant increase in proliferation as evidenced by CCK8, pluripotency markers OCT-4 tested by RT-PCR also showed increase. Above all, in LPS induced co-cultured inflame systerm, HUMSCs in extremely preterm were more capable to promote wound healing and tube formation in HUVEC cultures, they promoted TGFβ1 expression and inhibited IL6 expression.Conclusions:Our results suggest that HUMSCs from extremely preterm infants may be more suitable as candidates in cell therapy for the preterm infants.

Expression of cytokines and co-stimulatory molecules in the Toxoplasma gondii-infected dendritic cells of C57BL/6 and BALB/c mice.

Toxoplasma gondii is an intracellular protozoan parasite which can infect most warm-blooded animals and humans. Among the different mouse models, C57BL/6 mice are more susceptible to T. gondii infection compared to BALB/c mice, and this increased susceptibility has been attributed to various factors, including T-cell responses. Dendritic cells (DCs) are the most prominent type of antigen-presenting cells and regulate the host immune response, including the response of T-cells. However, differences in the DC responses of these mouse strains to T. gondii infection have yet to be characterized. In this study, we cultured bone marrow-derived DCs (BMDCs) from BALB/c and C57BL/6 mice. These cells were infected with T. gondii. The activation of the BMDCs was assessed based on the expression of cell surface markers and cytokines. In the BMDCs of both mouse strains, we detected significant increases in the expression of cell surface T-cell co-stimulatory molecules (major histocompatibility complex (MHC) II, CD40, CD80, and CD86) and cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-12p40, IL-1β, and IL-10) from 3 h post-T. gondii infection. The expression of MHC II, CD40, CD80, CD86, IFN-γ, IL-12p40, and IL-1β was significantly higher in the T. gondii-infected BMDCs obtained from the C57BL/6 mice than in those from the BALB/c mice. These findings indicate that differences in the activation status of the BMDCs in the BALB/c and C57BL/6 mice may account for their differential susceptibility to T. gondii.

Reduction in mRNA Expression of the Neutrophil Chemoattract Factor CXCL1 in Pseudomonas aeruginosa Treated Barth Syndrome B Lymphoblasts.

Barth Syndrome (BTHS) is a rare X-linked genetic disease caused by a mutation in the TAFAZZIN gene, which codes for the protein tafazzin involved in cardiolipin remodeling. Approximately 70% of patients with BTHS exhibit severe infections due to neutropenia. However, neutrophils from BTHS patients have been shown to exhibit normal phagocytosis and killing activity. B lymphocytes play a crucial role in the regulation of the immune system and, when activated, secrete cytokines known to attract neutrophils to sites of infection. We examined the expression of chemokine (C-X-C motif) ligand 1 (CXCL1), a known chemotactic for neutrophils, in Epstein-Barr virus transformed control and BTHS B lymphoblasts. Age-matched control and BTHS B lymphoblasts were incubated with Pseudomonas aeruginosa for 24 h and then cell viability, CD27+, CD24+, CD38+, CD138+ and PD1+ surface marker expression and CXCL1 mRNA expression determined. Cell viability was maintained in lymphoblasts incubated in a ratio of 50:1 bacteria:B cells. Surface marker expression was unaltered between control and BTHS B lymphoblasts. In contrast, CXCL1 mRNA expression was reduced approximately 70% (p < 0.05) in untreated BTHS B lymphoblasts compared to control and approximately 90% (p < 0.05) in bacterial treated BTHS B lymphoblasts compared to the control. Thus, naïve and bacterial-activated BTHS B lymphoblasts exhibit reduced mRNA expression of the neutrophil chemoattractant factor CXCL1. We suggest that impaired bacterial activation of B cells in some BTHS patients could influence neutrophil function via impairing neutrophil recruitment to sites of infection and this could potentially contribute to these infections.

Tualang and Kelulut Honey Reduced Lipopolysaccharides-stimulated Inflammatory Responses of Microglia

Microglial activity is crucial in maintaining the central nervous system (CNS) homeostasis. However, prolonged microglial activation have been implicated in the pathology of neurodegenerative diseases. Activated microglia will increase the production of inflammatory cytokines, reactive oxygen species (ROS) and alter their surface marker expression levels. This study used Malaysian honey, Tualang honey (TH), and Kelulut honey (KH) to determine lipopolysaccharide (LPS)-stimulated inflammatory responses of microglia. TH and KH at 0.1% were used in the current study as our findings showed no significant difference in the cell viability between BV2 cells treated with 0.1 % of TH and KH and control group. TH and KH reduced the ROS level significantly by 41.62±1.06% and 49.16±0.63%, respectively, and slightly reduced the expression of co-stimulatory molecules, CD40 and CD11b in LPS-activated BV2 cells. Our preliminary findings proposed an in-depth future study on the anti-inflammatory effect of TH and KH on microglial activation.

The internal dose makes the poison: higher internalization of polystyrene particles induce increased perturbation of macrophages.

Plastics are emerging pollutants of great concern. Macroplastics released in the environment degrade into microplastics and nanoplastics. Because of their small size, these micro and nano plastic particles can enter the food chain and contaminate humans with still unknown biological effects. Plastics being particulate pollutants, they are handled in the human body by scavenger cells such as macrophages, which are important players in the innate immune system. Using polystyrene as a model of micro and nanoplastics, with size ranging from under 100 nm to 6 microns, we have showed that although non-toxic, polystyrene nano and microbeads alter the normal functioning of macrophages in a size and dose-dependent manner. Alterations in the oxidative stress, lysosomal and mitochondrial functions were detected, as well as changes in the expression of various surface markers involved in the immune response such as CD11a/b, CD18, CD86, PD-L1, or CD204. For each beads size tested, the alterations were more pronounced for the cell subpopulation that had internalized the highest number of beads. Across beads sizes, the alterations were more pronounced for beads in the supra-micron range than for beads in the sub-micron range. Overall, this means that internalization of high doses of polystyrene favors the emergence of subpopulations of macrophages with an altered phenotype, which may not only be less efficient in their functions but also alter the fine balance of the innate immune system.

The Epigenetic Reader Protein SP140 Regulates Dendritic Cell Activation, Maturation and Tolerogenic Potential

SP140 is an epigenetic reader protein expressed predominantly in immune cells. GWAS studies have shown an association between SP140 single nucleotide polymorphisms (SNPs) and diverse autoimmune and inflammatory diseases, suggesting a possible pathogenic role for SP140 in immune-mediated diseases. We previously demonstrated that treatment of human macrophages with the novel selective inhibitor of the SP140 protein (GSK761) reduced the expression of endotoxin-induced cytokines, implicating a role of SP140 in the function of inflammatory macrophages. In this study, we investigated the effects of GSK761 on in vitro human dendritic cell (DC) differentiation and maturation, assessing the expression of cytokines and co-stimulatory molecules and their capacity to stimulate T-cell activation and induce phenotypic changes. In DCs, lipopolysaccharide (LPS) stimulation induced an increase in SP140 expression and its recruitment to transcription start sites (TSS) of pro-inflammatory cytokine genes. Moreover, LPS-induced cytokines such as TNF, IL-6, and IL-1β were reduced in GSK761- or SP140 siRNA- treated DCs. Although GSK761 did not significantly affect the expression of surface markers that define the differentiation of CD14+ monocytes into immature DCs (iDCs), subsequent maturation of iDCs to mature DCs was significantly inhibited. GSK761 strongly reduced expression of the maturation marker CD83, the co-stimulatory molecules CD80 and CD86, and the lipid-antigen presentation molecule CD1b. Finally, when the ability of DCs to stimulate recall T-cell responses by vaccine-specific T cells was assessed, T cells stimulated by GSK761-treated DCs showed reduced TBX21 and RORA expression and increased FOXP3 expression, indicating a preferential generation of regulatory T cells. Overall, this study suggests that SP140 inhibition enhances the tolerogenic properties of DCs, supporting the rationale of targeting SP140 in autoimmune and inflammatory diseases where DC-mediated inflammatory responses contribute to disease pathogenesis.

O163 The secretome of adipose-derived stem cells (ADSCS): exploring anti-fibrotic effects in skin fibrosis

Abstract Introduction As part of surgical reconstruction with autologous fat grafting, ADSCs are believed to play a pivotal role in the observed anti-fibrotic effect, however, the exact mechanisms remain unknown. Here, we explored the changes in the ADSC secretome after proinflammatory and profibrotic stimulation in-vitro to gain further mechanistic insights. Methods ADSCs were obtained from five non-obese patients (BMI&amp;lt;30) without systemic disease (n=5). ADSCs were exposed to either TGF-β1, IL-1β, hypoxia (1%O2) or 10% fetal bovine serum (FBS) for 24 hours. 0.1%FBS was used as control group. Samples were then analysed by RT-qPCR for nine target genes. Selected targets were validated in the secretome through ELISA. Further, expression of surface markers was explored by RT-qPCR. Results VEGF, FGF2, CSF1, and CCN5 were explored at protein level. VEGF secretion increased significantly in all treatment groups. IL-1β significantly increased the production of FGF2 at protein level (p=0.043). CSF1 was secreted in similar amounts across groups. CCN5 secretion varied little, although higher amounts were detected in the 10%FBS group. Hypoxia and IL-1β increased expression of ADSC markers (CD90, CD73, CD26), while TGF-β1 and 10%FBS decreased it. However, CD105 was upregulated in the 10%FBS group. Additionally, TIMP1, MMP2, APP, and OGN were explored. Conclusion ADSCs secreted VEGF, FGF2, CCN5, and CSF1 in an in-vitro fibrotic environment. Further investigation of these pathways may lead to the development of novel treatments for fibrotic diseases such as scleroderma and radiation-induced fibrosis.

Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques.

Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery.

NK cell defects: implication in acute myeloid leukemia.

Acute Myeloid Leukemia (AML) is a complex disease with rapid progression and poor/unsatisfactory outcomes. In the past few years, the focus has been on developing newer therapies for AML; however, relapse remains a significant problem. Natural Killer cells have strong anti-tumor potential against AML. This NK-mediated cytotoxicity is often restricted by cellular defects caused by disease-associated mechanisms, which can lead to disease progression. A stark feature of AML is the low/no expression of the cognate HLA ligands for the activating KIR receptors, due to which these tumor cells evade NK-mediated lysis. Recently, different Natural Killer cell therapies have been implicated in treating AML, such as the adoptive NK cell transfer, Chimeric antigen receptor-modified NK (CAR-NK) cell therapy, antibodies, cytokine, and drug treatment. However, the data available is scarce, and the outcomes vary between different transplant settings and different types of leukemia. Moreover, remission achieved by some of these therapies is only for a short time. In this mini-review, we will discuss the role of NK cell defects in AML progression, particularly the expression of different cell surface markers, the available NK cell therapies, and the results from various preclinical and clinical trials.

Harmonised culture procedures minimise but do not eliminate mesenchymal stromal cell donor and tissue variability in a decentralised multicentre manufacturing approach

BackgroundMesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A- to assess the impact on MSC characteristics when different laboratories, performed analysis on the same MSC material using harmonised culture conditions and B- to understand source-specific differences.MethodsThree independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue-specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B).ResultsBy implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro. In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs.ConclusionThese results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of harmonised culture procedures can reduce but do not eliminate inter-lab and operator differences.

Thresholding CITE-Seq-based Antibody Expression Data to Improve Cell Type Identification in scRNA-Seq

Abstract scRNA-seq is widely used to assess immune cell heterogeneity by measuring individual cell transcriptomes. Surface markers have been used for decades to type immune cells by flow and more recently mass cytometry. Expression of surface markers is poorly correlated with the mRNA levels of their encoding transcripts. CITE-Seq provides a systematic approach to analyze surface phenotypes along with cell transcriptomes by using oligonucleotide-tagged antibodies. Like flow cytometry, CITE-seq-based assessment of surface markers is complicated by non-specific binding. Here, we introduce and validate a systematic method of thresholding the levels of protein marker expression to address the issue of non-specific antibody binding. To remove the non-specific signal, a threshold value separating noise from expression for each surface marker is obtained by comparing the antibody signal in a cell type known to not express this surface marker. If no negative cell type is available, we use deconvolution of overlapping normal distributions to find the best separator in density plots (Ridgeline plots) by deconvolution (normalmixEM tool) of overlapping normal distributions. In total, 34 of 49 antibodies tested were reliably thresholded, thus effectively removing the noise from the signal. In a validation experiment testing 188 antibodies at 4 different concentrations, 124 were successfully thresholded, including all 34 mentioned above. Supported by the National Institutes of Health (NIH) P01 HL136275

Abstract A45: UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus

Abstract Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&amp;RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&amp;RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signature may be a viable therapeutic option. Citation Format: Juan M Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P Walsh, Jing Ma, Sherif Abdelhamed, Jeffery M Klco. UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A45.

Neutrophil Inhibitory Receptors Expression Is Distinct During Gram-Positive and -Negative Pneumonia and Affects Neutrophil Function

Abstract Rational: Despite effective antimicrobial therapy, morbidity and mortality from pneumonia remains high. Honing our understanding of immune dysfunction in pneumonia is now crucial for developing targeted therapeutics. Our objective is to understand how different lethal bacterial pneumonias affect neutrophil phenotype and function over time and identify regulators of neutrophil function. Methods: Mice were intratracheally instilled with E. colior S. pneumoniae(SP3) for 6, 24 or 48h and neutrophils were isolated from either bronchioloalveolar lavage fluid (BALF) or peripheral blood. Neutrophil surface marker expression (SME) was analyzed via 25-color panel run on a Cytek Aurora spectral flow cytometer (SFC). Data was analyzed in FlowJo and gated for live, single cell, CD45+Ly6G+ neutrophils, opt-SNE was run for data visualization, and Phenograph was run for unbiased clustering. For bacterial clearance assays, bone marrow neutrophils were isolated via Percoll, then incubated with blocking antibodies against inhibitor receptors (IRs) (PD-L1, SIRPα, VISTA, or CD200R), soluble ligands, or media only. Neutrophils were then cocultured with either SP3 or E. coli. Media from bacteria-neutrophil cultures was then plated on agar and the developing colonies were counted. Results and Conclusions: Using SFC, we identified pathogen- and timepoint-specific differences in SME of the IRs PD-L1, SIRPα, VISTA and CD200R. Modulating IR signaling by blocking or stimulating them impacts neutrophils’ ability to clear bacteria. In sum, our data suggests that neutrophils modulates their response to adapt to specific pathogens. IRs may play a role in regulating these dynamic neutrophil responses, making them a potential therapeutic target. NIH grants K08 HL130582 (KET) and R01 HL158732 (KET).

Genetic tracing of thymic T cell differentiation

Abstract Thymic T cell differentiation is controlled by stereotyped processes that remain obscure. Here we develop a novel mouse genetic tool to trace thymic T cells during T cell antigen receptor (TCR) rearrangement. Briefly, we insert inducible CreER into the Rag1 locus that is transiently expressed at early thymic T cell differentiation. In the presence of Rosa reporters, Rag1-CreER mice enable us to irreversibly label T cells between the double negative (DN) and double positive (DP) stages upon acute tamoxifen treatment. In the physiological setting and absence of the perturbations historically used to investigate thymic T cell differentiation, our genetic tracing in combination with flow cytometric measurement of cell fates and functional states maps the kinetics, stoichiometry, and cell fate determination of thymic T cells. We further integrate genetic tracing experiments with unbiased single cell RNA sequencing, TCR sequencing, and the expression of cell surface markers, linking thymic T cell differentiation stages to dynamic gene expression. In particular, we show that regulatory T cells acquire CD73 expression over time as a potential consequence of persistent TCR signaling and expression of lineage determinant Foxp3, marking a maturation process during lineage commitment and functional adaptation. Taken together, our integrated genetic tracing of thymic T cells resolves complex differentiation processes coupled with the alterations of gene expression, selection, and cell fate determination. Our results also serve as a foundation for further exploration of the thymic T cell states, developmental pathways, drivers, and modulators involved in these crucial biological processes. R21 AI146614