Common Lymphoid Progenitors Research Articles

Inhibition of B cell activation by Ibrutinib improves cardiac function in experimental myocardial infarction in mice

Abstract Background Bruton's tyrosine kinase (BTK) is a key downstream mediator of B cell receptor (BCR) signaling. Upon BCR cross-linking, phosphorylated BTK modulates B cell activation, proliferation, and differentiation. Ibrutinib, which is clinically used in B-cell malignancies, irreversibly binds to the BTK kinase domain and blunts downstream BTK signaling. Purpose Here, we aimed to monitor B cell dynamics and activation in experimental myocardial infarction (MI) in mice, and evaluate the effect of B cell inhibition by Ibrutinib in cardiac function. Methods MI was induced in male 10-week-old C57BL/6 mice by permanent LAD ligation and Ibrutinib (25 mg/kg/day) was given in the drinking water. After 3, 7, 14, and 28 days, single cell suspensions were prepared for flow cytometry from peripheral blood, spleen, mediastinal lymph nodes (MLNs), and the bone marrow, as well as from the remote and infarcted myocardium. Total B cells and B cell subsets were quantified in the different locations by flow cytometry and B cell activation was monitored by BTK551 phosphorylation. After 28 days, cardiac function was evaluated by echocardiography. Results We found that total B cell numbers decrease in blood and spleen, and increase in MLNs, while plasma cells and plasmablasts increased in the infarcted heart on day 7 and 14 after MI. In the bone marrow, hematopoietic stem cells (HSC) increase after MI, while common lymphoid progenitors (CLP) and early-stage B cell progenitors (pre-pro and pro B cells) peak at day 7 after MI. The later-stage B cell progenitors (pre and immature B cells) showed a biphasic response, with an early decrease after 7 days and a later increase on day 14. Compared to the control group (vehicle), Ibrutinib treatment decreased B cell numbers in MLN and spleen by 27% and 14%, respectively, as well as decreased mature B cells by 28% in the bone marrow on day 28 after MI. Both BTK expression and phosphorylation in pro-B cells were inhibited by Ibrutinib by 8% and 15%, respectively. The inhibition of BTK phosphorylation was also evident in MLNs and spleen. Notably, Ibrutinib-treated mice showed an improved cardiac function on day 28 after MI, with stroke volume (SV) elevated by 41%, left ventricular ejection fraction (LVEF) elevated from 20% to 36% (p < 0.05), and fractional shortening (FS) elevated from 8.8% to 17.59% (p < 0.05). Conclusion Experimental MI leads to B cell lymphopoiesis in the bone marrow, mobilization into the infarcted myocardium, and activation in peripheral lymphoid organs. Ibrutinib treatment inhibits B cell development in the bone marrow, as well as B cell activation in peripheral lymphoid organs, and improves cardiac function post MI. Our data provides a meaningful insight for targeting B cells for the clinical treatment of patients with MI.Figure

Myeloid deficiency of heparan sulfate 6-O-endosulfatases impairs bone marrow hematopoiesis

The heparan sulfate (HS) 6-O-endosulfatases or the Sulfs (Sulf1 and Sulf2) are the only known enzymes that can modify HS sulfation status extracellularly and have been shown to regulate diverse biological processes. The role of the Sulfs in bone marrow (BM) hematopoiesis is not known. In this study, we generated a novel mouse line with myeloid-specific deletion of the Sulfs by crossing Sulf1/2 double floxed mice with the LysM-cre line. The LysM-Sulf knockout (KO) male mice exhibited age-dependent expansion of hematopoietic stem cells and the granulocyte-monocyte lineages in the BM, whereas common lymphoid progenitors and B lymphocyte populations were significantly reduced. Although megakaryocytic and erythroid progenitors were not reduced in the BM, the LysM-Sulf KO males suffered age-dependent reduction of red blood cells (RBCs) and platelets in the peripheral blood, suggesting that the production of RBCs and platelets was arrested at later stages. In addition, LysM-Sulf KO males displayed progressive splenomegaly with extramedullary hematopoiesis. Compared to males, LysM-Sulf KO females exhibited a much-reduced phenotype, and ovariectomy had little effect. Mechanistically, reduced TGF-β/Smad2 but enhanced p53/p21 signaling were observed in male but not female LysM-Sulf KO mice. Finally, HS disaccharide analysis via LC-MS/MS revealed increased HS 6-O-sulfation in the BM from both male and female LysM-Sulf KO mice, however, the distribution of 6-O-sulfated motifs were different between the sexes with compensatory increase in Sulf1 expression observed only in LysM-Sulf KO females. In conclusion, our study reveals that myeloid deficiency of the Sulfs leads to multilineage abnormalities in BM hematopoiesis in an age- and sex-dependent manner.

Azacitidine and cytarabine induce sustained lymphopenia with abnormal differentiation of common lymphoid progenitors and prolonged suppression of Dnmt3a and Dnmt3b expression in mice.

Myelosuppression is a major side effect of chemotherapy. Although decreased blood cells are restored with the recovery of bone marrow cells, insufficient recovery of decreased lymphocytes was observed in mice given azacitidine (AZA), a DNA methyltransferase (DNMT) inhibitor, even following the restoration of bone marrow cells. To understand the mechanisms behind this sustained lymphopenia, we examined AZA's impact on the hematopoietic progenitor cells and the expression of Dnmts and differentiation-related genes. An antimetabolite of cytidine analog, cytarabine (Ara-C), was used as a reference compound. Decreases in almost all blood parameters and common lymphoid progenitors (CLPs) and the downregulation of Dnmts and differentiation-related genes in Lineage-Sca-1+c-kit+ (LSK) cells were observed in mice administered AZA or Ara-C for 7 d. In the posttreatment observation, all parameters, except for lymphocytes and monocytes, exhibited recovery within 3 wk after the final dosing in both treated groups. However, no recovery from the decreases in lymphocytes, especially B cells, and monocytes occurred even after 5 wk. The number of CLPs was elevated after 3 wk. There was a tendency toward recovery from the decreased expression of Dnmt1 and differentiation-related genes, but the expression levels of Dnmt3a and Dnmt3b did not fully recover even 5 wk after the final dosing. Taken together, the findings revealed that the mechanism of sustained lymphopenia observed in mice treated with AZA or Ara-C is associated, at least in part, with the abnormal differentiation of CLPs into B cells accompanied by the prolonged suppression of Dnmt3a and Dnmt3b expression on LSK cells.

Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Ferulic Acid Alleviates Radiation-Induced Immune Damage by Acting on JAK/STAT Signaling Pathway.

The disruption of hematopoietic and immune functions is a significant consequence of the long-term effects of radiation exposure. This study investigated the potential mechanisms by which ferulic acid (FA) acts as a radioprotective agent in mitigating radiation-induced immune damage. C57BL/6J mice were exposed to a dose of 6.0 Gy of 60Co γ irradiation. FA was administered at doses of 25, 50, and 100 mg/kg/d for 7 days before and 30 days following irradiation. We evaluated changes in peripheral blood cells, T and B lymphocytes, natural killer cells in the spleen, and hematopoietic stem/progenitor cells in the bone marrow (BM). Whole-genome transcriptome sequencing of BM was performed to explore potential mechanisms. FA administration resulted in a significant reduction in malonaldehyde levels (p < 0.0001), an increase in catalase and beta-nicotinamide adenine dinucleotide levels in serum (p < 0.05), and enhanced multipotent progenitors (p < 0.01) and common lymphoid progenitors (p < 0.05) in the BM. Additionally, there was an elevation in white blood cell levels, red blood cell levels, and hemoglobin levels in peripheral blood (p < 0.01). Transcriptome analysis indicated that FA reversed the radiation-induced expression of genes related to immunity and inflammation. Enzyme-linked immunosorbent assay experiments further demonstrated that FA reduced interleukin-6 levels in the BM and decreased JAK1, JAK2, and STAT3 protein content (p < 0.01). In conclusion, FA might mitigate hematopoietic and immune damage by modulating the JAK/STAT signaling pathway.

Abstract P128: Endothelial Nitric Oxide Determines Hematopoietic Cell Fate and Osteoclastogenesis By Modulating CSF1 During Hypertension

Introduction: Loss of endothelial nitric oxide (NO) is common in hypertension (HTN). The bone marrow niche contains endothelial cells; however, how endothelial NO affects hematopoiesis and bone in HTN remains undefined. Hypothesis: We hypothesized that NO loss in HTN drives hematopoiesis in the bone marrow, later favoring osteoclastogenesis and bone resorption. Methods and Results: By microcomputed tomography, eNOS deficient mice (eNOS -/- ) had reduced femoral cortical area (Figure 1A) and trabecular bone volume fraction (Figure 1B) compared to wildtype mice (WT). Flow cytometry showed that granulocyte-monocyte (Figure 1C) and common lymphoid progenitors (Figure 1D) were markedly higher in eNOS -/- than in WT. mRNA analysis revealed loss of endothelial NO increased expression of cathepsin K (Ctsk), colony stimulating factor-1 receptor (CSF1R), and tartrate resistant acid phosphatase (TRAP) in the bone marrow. We exposed murine bone-marrow endothelial cells (BMECs) to 5% (normotension), 5% with L-NAME, or 10% (HTN) cyclic strain. Compared to 5% strain, BMECs exposed to L-NAME or 10% strain exhibited markedly increased CSF1 production, an essential cytokine for myelopoiesis and osteoclastogenesis (Figure 1E and F). In coculture experiments, we also found that macrophages cultured with BMECs undergoing 5% strain with L-NAME or 10% strain exhibited higher expression of Ctsk, CSF1R, and TRAP, indicating osteoclast formation. Conclusion: Endothelial NO likely inhibits osteoclastogenesis and bone resorption. The loss of NO through mechanical stretch or inhibiting eNOS increases CSF1 production and hematopoiesis. This shows a potential mechanism by which NO loss leads to pathological hematopoiesis and bone resorption during HTN.

Tetrahydroxy stilbene glucoside rejuvenates aging hematopoietic stem cells with predilection for lymphoid differentiation via AMPK and Tet2

IntroductionAging of hematopoietic stem cells (HSCs) has emerged as an important challenge to human health. Recent advances have raised the prospect of rejuvenating aging HSCs via specific medical interventions, including pharmacological treatments. Nonetheless, efforts to develop such drugs are still in infancy until now. ObjectivesWe aimed to screen the prospective agents that can rejuvenate aging HSCs and explore the potential mechanisms. MethodsWe screened a set of natural anti-aging compounds through oral administration to sub-lethally irradiated mice, and identified 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) as a potent rejuvenating agent for aging HSCs. Then naturally aged mice were used for the follow-up assessment to determine the HSC rejuvenating potential of TSG. Finally, based on the transcriptome and DNA methylation analysis, we validated the role of the AMP-activated protein kinase (AMPK)-ten-eleven-translocation 2 (Tet2) axis (the AMPK-Tet2 axis) as the underlying mechanisms of TSG for ameliorating HSCs aging. ResultsTSG treatment not only significantly increased the absolute number of common lymphoid progenitors (CLPs) along with B lymphocytes, but also boosted the HSCs/CLPs repopulation potential of aging mice. Further elaborated mechanism research demonstrated that TSG supplementation restored the stemness of aging HSCs, as well as promoted an epigenetic reprograming that was associated with an improved regenerative capacity and an increased rate of lymphopoiesis. Such effects were diminished when the mice were co-treated with an AMPK inhibitor, or when it was performed in Tet2 knockout mice as well as senescent cells assay. ConclusionTSG is effective in rejuvenating aging HSCs by modulating the AMPK- Tet2 axis and thus represents a potential candidate for developing effective HSC rejuvenating therapies.

An anoikis-related lncRNA signature may predict the prognosis, immune infiltration, and drug sensitivity in esophageal cancer

BackgroundEsophageal cancer (EC) is a prevalent malignancy with heterogeneous outcomes. This study explores the significance of anoikis-related long non-coding RNAs (lncRNAs) in EC, aiming to unravel their molecular roles and clinical implications. MethodsTranscriptome and clinical data were obtained from TCGA database for EC samples. We identified anoikis-related genes and lncRNAs by Pearson correlation analysis. The risk score model hinged on prognostic lncRNAs filtered from multiple steps. Risk scores were calculated using the derived formula, and categorized patients into low- and high-risk groups. Model robustness was assessed through Kaplan-Meier (KM) survival analysis and Receiver Operating Characteristic (ROC) curve, with clinical utility achieved via a constructed nomogram. We also explored the interplay between the risk score and immune cell infiltration, and investigated drug sensitivity. ResultsWe identified 2365 anoikis-related lncRNAs through co-expression analysis, including 1415 significant lncRNAs differentially expressed between normal and tumor samples. A risk score model was constructed from ten prognostic lncRNAs. The risk score model effectively stratified patients based on the median score, and its predictive capacity was validated through KM survival, ROC curve analyses, and the external GSE53622 dataset. The nomogram provided a practical tool for individualized prognosis evaluation. We unveiled significant correlations between specific immune cell subsets and the risk score. Eosinophils and common lymphoid progenitors exhibited positive associations, while endothelial cells and myeloid dendritic cells showed negative correlations. Drug sensitivity analysis revealed potential sensitive drugs for EC treatment that aligned with the risk subgroups. ConclusionThis study established an anoikis-related lncRNAs risk score model that may predict the prognosis, immune infiltration, and drug sensitivity in EC, in hope of facilitating tailored patient management.

Differential Gene Expression Profiling during Avian Immune Organ Development: Insights from Thymus and Bursa of Fabricius Transcriptome Analysis

Pluripotent hematopoietic stem cells undergo maturation to regenerate blood cells. T-cell and B-cell maturation from common lymphoid progenitors (CLPs) is regulated by distinct transcription factors and signaling pathways. Notch signaling, GATA3, and TCF1 drive T-cell fate, while EBF1, PU.1, E2A, Pax5, TCF1, and Foxo1 regulate B-cell differentiation. Alterations in these factors can cause lineage deviations. Gene expression profiling of the chick thymus and bursa reveals dynamic expression patterns of transcription factors, cytokines, and signal molecules. Notch1 and Dll1 expression that increase during later stages indicate the ongoing role of Notch signaling in T-cell lineage maintenance. The current study aimed to identify differentially expressed genes during the development of the avian immune organs, focusing on the thymus and bursa using 24 Ross 308 avian breed. The mRNA libraries from these organs were analyzed using quantitative Real-time PCR analysis at six time points spanning the embryonic ages (days 15 and 18) and post-hatch age (days 3, 7, 14, and 28). The data for the gene expression indicated significant variations across different stages of immune organ development. Differential gene expression was observed between sorted T and B-cells, with GATA3, CD3e, CD4, and Ptprc showing higher expression in the T-cell population, and Pax5 and CD81 exhibiting higher expression in the B-cell population. Notably, ENO1 and IRF4 showed higher expression in T-cells at E15 and B-cells at E18. The study highlights the importance of regulatory factors and genes in maintaining cellular identity, furthers the understanding of avian immunology, and has the potential for improving poultry health and studying immune-related diseases in humans. These findings pave the way for further research on the role of biochemical components under important disease conditions in avian immunology and their potential applications for poultry health. Keywords: Avian lymphocyte, B-cell maturation, Gene expression, Regulatory elements, T-cell

Abstract 1580: Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancers with a survival rate of less than 12% at 5 years. It is estimated that around 14% of human PDAC contain mutations in genes involved in DNA Damage Repair (DDR), including BRCA1/2, ATM, and others. Additionally, PDAC has an extensive network of extracellular matrix components surrounding the tumor in the stroma, such as cancer-associated fibroblasts, which promote cancer cell growth. The interactions of the tumor with its tumor microenvironment and stroma can bring vulnerabilities that could be targeted if identified. New therapeutic strategies to those targets can be developed for specifically PDAC with DDR mutations. Using publicly available RNA-seq dataset of PDAC patients from The Cancer Genome Atlas (TCGA), I computationally estimated the infiltration of immune and stromal cell types into the tumor. From the analyses, I found that there was a significant difference in the migration of Common Lymphoid Progenitor (CLP) cells into PDAC with mutations to DDR genes. CLP cells usually divide into T-cells, B-cells, and NK cells, suggesting that DDR mutations could cause a more immunogenic environment. There was also a decrease in the infiltration of stromal elements, mainly fibroblasts, into PDAC with mutations to DDR genes. In the experiment I am currently working on, I hypothesize there are also in vivo differences in the infiltration rates of certain immune cell types and fibroblasts into BRCA2-mutated PDAC, since BRCA2 is the most commonly mutated DDR gene in PDAC. For my current experimental design, the mice in the experimental group will be orthotopically implanted with PDAC with Brca2 knockout. The control group will have mice orthotopically implanted with PDAC without Brca2 knockout. After letting the tumors grow for 3 weeks, the mice will be sacrificed and the pancreatic tumors will be collected and stained through trichrome staining. Trichrome staining probes for the stromal cancer-associated fibroblasts. Furthermore, immunohistochemistry (IHC) staining will be done to look for specific populations of cells, such as specific types of fibroblasts, effective and immunosuppressive immune cells including T-cells, B-cells, and NK cells. The aim is to see whether the computational findings in regards to the tumor microenvironment are also reflected in vivo. I will have the data from this experiment by the time of the conference in April 2024. Overall, my project is about investigating the tumor microenvironment and fibroblast abundance in PDAC with BRCA2 knockout mice, using computational data, IHC staining and trichrome staining. Citation Format: Fatimah Al-Musawi, Chang-il Hwang. Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1580.

Acute malaria suppresses the B lymphocytic niche in the bone marrow through the alteration of CXCL12-abundant reticular cells.

Bone marrow is a dynamic organ composed of stem cells that constantly receive signals from stromal cells and other hematopoietic cells in the niches of the bone marrow to maintain hematopoiesis and generate immune cells. Perturbation of the bone marrow microenvironment by infection and inflammation affects hematopoiesis and may affect immune cell development. Little is known about the effect of malaria on the bone marrow stromal cells that govern the hematopoietic stem cell (HSC) niche. In this study, we demonstrate that the mesenchymal stromal CXCL12-abundant reticular (CAR) cell population is reduced during acute malaria infection. The reduction of CXCL12 and IL-7 signals in the bone marrow impairs the lymphopoietic niche, leading to the depletion of common lymphoid progenitors, B cell progenitors and mature B cells, including plasma cells in the bone marrow. We found that IFNγ is responsible for the upregulation of Sca1 on CAR cells, yet the decline in CAR cell and B cell populations in the bone marrow is IFNγ-independent. In contrast to the decline in B cell populations, HSCs and multipotent progenitors increased with expansion of myelopoiesis and erythropoiesis, indicating a bias in the differentiation of multipotent progenitors during malaria infection. These findings suggest that malaria may affect host immunity by modulating the bone marrow niche.

Low expression of GRM4 is associated with poor prognosis and tumor immune infiltration in glioma

Introduction The metabotropic glutamate receptor 4 (mGlu4, GRM4) exhibits significant expression within the central nervous system (CNS) and has been implicated to be correlated with a poor prognosis. Objective This study was aimed to elucidate the relationship between the expression profile of GRM4 and the prognosis of glioma patients. Methods RNA-sequencing datasets from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and China Glioma Genome Atlas (CGGA) repositories were used to evaluate the potential relationship. The value of clinical prognostic about GRM4 was assessed using clinical survival data from CGGA and TCGA. The GEPIA database was used to select genes like GRM4. PPI network was constructed by the database of (STRING), GO and KEGG analyses were performed. TargetScan, TarBase, miRDB, and starBase were used to explore miRNAs that could regulate GRM4 expression. EWAS Data Hub, MethSurv, and MEXPRESS were used for the analysis and relationship between DNA methylation and GRM4 expression and prognosis in glioma. TIMER2.0 and CAMOIP databases were used to assess the association between immune cell infiltration and GRM4. Human GBM cell lines were used to validate the function of GRM4. Results Our study shows that GRM4 is under expressed among gliomas and accompanied by poorer OS. Multivariate analysis showed that low mRNA expression of GRM4 was an independent factor of prognostic for shorter OS in all glioma patients. MiR-1262 affects the malignant phenotype of gliomas through GRM4. Methylation of DNA plays an important role in the instruction of GRM4 expression, the methylation level of GRM4 in glioma tissue is higher in comparison to normal tissue, and the higher methylation level was accompanied with the worse prognosis. Further analysis showed that GRM4 mRNA expression in GBM linked negatively with common lymphoid progenitor, Macrophage M1, Macrophage, and T cell CD4+ Th2, but not with the tumor purity. Overexpression of GRM4 prevents the migration of human GBM cell lines in vitro. Conclusion GRM4 may have a substantial impact on the infiltration of immune cells and serve as a valuable prognostic biomarker in gliomas.

PCMT1 confirmed as a pan-cancer immune biomarker and a contributor to breast cancer metastasis.

Protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT, gene name PCMT1) is an enzyme that repairs proteins with altered aspartate residues by methylation, restoring their normal structure and function. This study conducted a comprehensive analysis of PCMT1 in pan-cancer. The Cancer Genome Atlas, Human Protein Atlas website, and the Genotype-Tissue Expression were utilized in analysis of PCMT1 expression. We examined the association between PCMT1 expression and various factors, including gene modifications, DNA methylation, immune cell infiltration, immunological checkpoints, drug susceptibility, tumor mutation burden (TMB), and microsatellite instability (MSI). Enrichment analyses determined the potential biological roles and pathways involving PCMT1. Our focus then shifted to the role of PCMT1 in breast invasive carcinoma (BRCA). We found that PCMT1 expression was aberrant in many tumors and significantly influenced the prognosis across several cancer types. Gene alterations in PCMT1 predominantly involved deep deletions and amplifications. A negative correlation was observed between DNA methylation and PCMT1 expression across all studied cancer types except thyroid carcinoma PCMT1 exhibited positive correlations with common lymphoid progenitor and CD4(+) T helper 2 cells, whereas it was inversely correlated with central and effector memory T cells, memory CD8(+) T cells, and CD4(+) T helper 1 cells. In many cancer types, PCMT1 expression closely correlated with immunological checkpoint inhibitors, TMB, and MSI. It was also significantly linked to pathways involved in epithelial-mesenchymal transition (EMT), highlighting its role in cancer metastasis. PCMT1 emerged as a significant predictor of breast cancer progression. In vitro experiments demonstrated that reducing PCMT1 expression decreased BRCA cell migration and invasiveness. Additionally, animal studies confirmed that inhibition of PCMT1 slowed tumor growth.

A Pyroptosis-Related Gene Signature Predicts Prognosis and Tumor Immune Microenvironment in Colorectal Cancer.

Pyroptosis is a programmed cell death, which garners increasing attention by relating to immune and therapy response. However, the role of pyroptosis in colorectal cancer (CRC) remains unclear. Our study mainly to explore the role of pyroptosis in CRC. The mRNA expression data and corresponding clinical information of CRC patients were achieved from The Cancer Genome Atlas (TCGA). Pyroptosis-related genes (PRGs) were identified using DESeq2 R package and biological function was analyzed using cluster Profiler R package. A PRGs-based prognosis model was constructed by a univariate Cox and LASSO regression analyses. Then, the affecting of risk signature to clinicopathological characteristics, immune status and infiltrated immune cells, immune checkpoint and chemotherapy sensitivity was analyzed. qRT-PCR and IHC were performed for the expression level of PRGs. Moreover, a nomogram predict model was constructed. Total 57 PRGs were identified between 500 CRC samples and 44 normal samples. Those PRGs mainly enriched in immune-related and pyroptosis-related pathways. GABRD, NADK, TMEM240, RER1, AGRN, UBE2J2, CALML6, PLCH2, TMEM88B have been identified as gene signature and a prognostic model was constructed and validated. CRC patients with high-risk score showed poor survival, high TMB score, high proportion of CD4 + memory T cells, common lymphoid progenitors, cancer associated fibroblasts, mast cells, and neutrophils. The immune checkpoint related genes, CD160, CD200R1, CD244, CD28, CD40LG, CD44, CD48, CD80, CD86, HHLA2, ICOS, IDO1, TIGIT, TNFRSF25, TNFRSF4, TNFRSF9, TNFSF15, TNFSF18 also increased in high-risk score group. CRC patients with high-risk score more sensitive to docetaxel and rapamycin but resistance to gemcitabine and mitomycin. Moreover, a predictive nomogram for 1-, 3-, 5-year for CRC patients was established and validated. In the study, a PRGs-based prognostic model and a predictive model were constructed. These models are effective and robust in prediction the 1-, 3-, and 5-year survival of CRC patients.

An Investigation of the Inflammatory Landscape in the Brain and Bone Marrow of the APP/PS1 Mouse.

The APP/PS1 mouse model recapitulates pathology of human Alzheimer's disease (AD). While amyloid-β peptide deposition and neurodegeneration are features of AD, the pathology may involve inflammation and impaired vascular regeneration. This study evaluated inflammatory environments in the brain and bone marrow (BM), and the impact on brain microvascular density. BM and frontal cortex from male nine-month-old APP/PS1 or the control C57Bl6/j mice were studied. Vascular density and inflammatory cells were evaluated in the sections of frontal cortex by immunohistochemistry. Different subsets of hematopoietic stem/progenitor cells (BM) and monocyte-macrophages were characterized by flow cytometry and by clonogenic assays. Myelopoietic or inflammatory factors were evaluated by real-time RT-PCR or by western blotting. CD34+ or CD31+ vascular structures were lower (p < 0.01, n = 6) in the frontal cortex that was associated with decreased number of Lin-Sca-1+cKit+ vasculogenic progenitor cells in the BM and circulation (p < 0.02, n = 6) compared to the control. Multipotent progenitor cells MPP4, common lymphoid, common myeloid and myeloid progenitor cells were higher in the APP/PS1-BM compared to the control, which agreed with increased numbers of monocytes and pro-inflammatory macrophages. The expression of pro-myelopoietic factors and alarmins was higher in the APP/PS1 BM-HSPCs or in the BM-supernatants compared to the control. Frontal cortices of APP/PS1 mice showed higher number of pro-inflammatory macrophages (CD11b+F4/80+ or CD80+) and microglia (OX42+Iba1+). These findings show that AD pathology in APP/PS1 mice is associated with upregulated myelopoiesis, which contributes to the brain inflammation and decreased vascularity.

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

PELI2 regulates early B-cell progenitor differentiation and related leukemia via the IL-7R expression.

Little is known about the transition mechanisms that govern early lymphoid lineage progenitors from common lymphoid progenitors (CLP). Pellino2 (PELI2) is a newly discovered E3 ubiquitin ligase, which plays important roles in inflammation and the immune system. However, the physiological and molecular roles of PELI2 in the differentiation of immune cells are largely unknown. Here, by using a conditional knockout mouse model, we demonstrated that PELI2 is required for early B-cell development and stressed hematopoiesis. PELI2 interacted with and stabilized PU.1 via K63-polyubiquitination to regulate IL-7R expression. The defects of B-cell development induced by PELI2 deletion were restored by overexpression of PU.1. Similarly, PELI2 promoted TCF3 protein stability via K63-polyubiquitination to regulate IL-7R expression, which is required for the proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. These results underscore the significance of PELI2 in both normal B lymphopoiesis and malignant B-cell acute lymphoblastic leukemia via the regulation of IL-7R expression, providing a potential therapeutic approach for BCP-ALL.

Loss of SMARCA4 Leads to an Impaired Hematopoiesis in Mice

SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a) is the central ATPase containing enzyme in the SWI/SNF- (switch/sucrose non-fermentable) complex, which regulates chromatin accessibility and thereby influences gene transcription. By acting as a transcriptional activator or repressor in different cell types, SMARCA4 and the SWI/SNF complex have various functions. Our aim was to understand the function of SMARCA4 in healthy hematopoiesis in order to then apply our findings to different hematologic diseases in a second step. To investigate the role of SMARCA4 in haematopoiesis and hematopoietic stem cells, we created a transgenic Smarca4 knockout mouse model. An inducible knockout system was chosen since early embryonic knockout of SMARCA4, which is crucially involved in embryonic development leads to a lethal phenotype. The Cre/loxP system was used to induce homozygous Smarca4 knockout under the control of the hematopoiesis-specific promotor Mx-1. We then performed blood counts, bone marrow smears and RNA sequencing, including analysis of RNA signature of the different cell types in the bone marrow to decipher the composition of the bone marrow of Smarca 4 deficient mice. Blood count analysis showed that Smarca4 knockout mice have significantly lower leukocyte, reticulocyte, and thrombocyte counts. In addition, bone marrow showed lower colony forming capacity in colony formation assays compared to healthy cre-negative control mice. Bone marrow smears displayed aplasia of all cell lines in Smarca4 knockout mice with additional signs of dysplasia as hypolobulatedmegakaryocytes or nuclear blasts. RNA-sequencing revealed 749 upregulated and 520 downregulated genes with a log fold change ≥ +/- 1 in the bone marrow of Smarca4 knockout mice compared to cre-negative control mice. Relevant over represented pathways according to REACTOME data base included genes of generation of second messenger molecules, cell surface interactions at the vascular wall, and immunoregulatory interactions between lymphoid and non-lymphoid cells. Through cell type deconvolution (CIBERSORTx) of the bulk microarray gene expression data we estimated abundances for different cell types. Results revealed a decrease in granulocyte and macrophage progenitors (statistically significant) as well as in eosinophil progenitor cells, and in erythroid pre-colony forming units. Interestingly, both common myeloid and common lymphoid progenitors were increased. It seems as if the progenitor cells accumulate and take a different path when Smarca4 is knocked out in these cells (figure 2). We conclude that SMARCA4 is crucial for cell growth and differentiation of progenitor cells in the bone marrow during hematopoiesis since a Smarca4 knockout leads to differentiation arrest and accumulation of distinct progenitor cells and pancytopenia in peripheral blood. Figure 1: Abundances for 23 cell populations in Smarca4 knockout (KO) and wildtype (WT) samples were estimated using CIBERSORTx. Bubble plot showing Log2 fold changes of average abundances in KO vs WT with accumulation of distinct progenitor cells in the bone marrow of Smarca4 deficient mice. As underlying signature-matrix the haemopedia gene set was used. Bubbles with black borders indicate significance of difference between samples.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Interleukin-17A Directly Signals to Bone Marrow-Derived Hematopoietic Stem and Progenitor Cells to Promote Their Expansion and Differentiation in Vitro

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.