Lymph node macrophages drive innate immune responses to enhance the anti-tumor efficacy of mRNA vaccines.

mRNA vaccines are promising for cancer treatment. Efficient delivery of mRNAs encoding tumor antigens to antigen-presenting cells (APCs) is critical to elicit anti-tumor immunity. Herein, we identified a novel lipid nanoparticle (LNP) formulation, L17-F05, for mRNA vaccines by screening 34 ionizable lipids and 28 LNP formulations using human primary APCs. Subcutaneous delivery of L17-F05 mRNA vaccine encoding Gp100 and Trp2 inhibited tumor growth and prolonged the survival of mice bearing B16F10 melanoma. L17-F05 efficiently delivered mRNAs to conventional dendritic cells (cDCs) and macrophages in draining lymph nodes (dLNs). cDCs functioned as the main APCs by presenting antigens along with enhanced expression of co-stimulatory molecules. Macrophages triggered innate immune responses centered on type-I interferon (IFN-I) in dLNs. Lymph node (LN) macrophage depletion attenuated APC maturation and anti-tumor activity of L17-F05 mRNA vaccines. Loss-of-function studies revealed that L17-F05 works as a self-adjuvant by activating the stimulator of interferon genes (STING) pathway in macrophages. Collectively, the self-adjuvanticity of L17-F05 triggered innate immune responses in LN macrophages via the STING-IFN-I pathway, contributing to APC maturation and potent anti-tumor activity of L17-F05 mRNA vaccines. Our findings provide strategies for further optimization of mRNA vaccines based on the innate immune response driven by LN macrophages.

The Significance of SPP1 in Lung Cancers and Its Impact as a Marker for Protumor Tumor-Associated Macrophages.

Macrophages are a representative cell type in the tumor microenvironment. Macrophages that infiltrate the cancer microenvironment are referred to as tumor-associated macrophages (TAMs). TAMs exhibit protumor functions related to invasion, metastasis, and immunosuppression, and an increased density of TAMs is associated with a poor clinical course in many cancers. Phosphoprotein 1 (SPP1), also known as osteopontin, is a multifunctional secreted phosphorylated glycoprotein. Although SPP1 is produced in a variety of organs, at the cellular level, it is expressed on only a few cell types, such as osteoblasts, fibroblasts, macrophages, dendritic cells, lymphoid cells, and mononuclear cells. SPP1 is also expressed by cancer cells, and previous studies have demonstrated correlations between levels of circulating SPP1 and/or increased SPP1 expression on tumor cells and poor prognosis in many types of cancer. We recently revealed that SPP1 expression on TAMs is correlated with poor prognosis and chemoresistance in lung adenocarcinoma. In this review, we summarize the significance of TAMs in lung cancers and discuss the importance of SPP1 as a new marker for the protumor subpopulation of monocyte-derived TAMs in lung adenocarcinoma. Several studies have shown that the SPP1/CD44 axis contribute to cancer chemoresistance in solid cancers, so the SPP1/CD44 axis may represent one of the most critical mechanisms for cell-to-cell communication between cancer cells and TAMs.

Exposure to microbial products followed by loss of Tet2 promotes myelodysplastic syndrome via remodeling HSCs.

Aberrant innate immune signaling in myelodysplastic syndrome (MDS) hematopoietic stem/progenitor cells (HSPCs) has been implicated as a driver of the development of MDS. We herein demonstrated that a prior stimulation with bacterial and viral products followed by loss of the Tet2 gene facilitated the development of MDS via up-regulating the target genes of the Elf1 transcription factor and remodeling the epigenome in hematopoietic stem cells (HSCs) in a manner that was dependent on Polo-like kinases (Plk) downstream of Tlr3/4-Trif signaling but did not increase genomic mutations. The pharmacological inhibition of Plk function or the knockdown of Elf1 expression was sufficient to prevent the epigenetic remodeling in HSCs and diminish the enhanced clonogenicity and the impaired erythropoiesis. Moreover, this Elf1-target signature was significantly enriched in MDS HSPCs in humans. Therefore, prior infection stress and the acquisition of a driver mutation remodeled the transcriptional and epigenetic landscapes and cellular functions in HSCs via the Trif-Plk-Elf1 axis, which promoted the development of MDS.

Involvement of protumor macrophages in breast cancer progression and characterization of macrophage phenotypes.

Tumor-associated macrophages (TAMs) are the most prominent immune cells in the breast cancer microenvironment, and the protumor functions of TAMs are thought to affect cancer progression and resistance to anticancer therapy. Numerous studies using human breast cancer samples, cell lines, and murine breast cancer models have revealed details of the mechanisms by which the protumor functions of TAMs are activated. Recent advances have highlighted the significant involvement of TAMs in the resistance of breast cancer cells to immunotherapy. Tumor-associated macrophages express a number of immunosuppressive genes, and single-cell sequence analyses of human and murine cancer samples have helped elucidate the mechanism of TAM-induced immunosuppression. As TAMs are considered suitable targets for anticancer therapies, we summarized the protumor functions of TAMs and the potential of anticancer therapies targeting TAMs, with a focus on breast cancer research.

Multivalent mannose-conjugated siRNA causes robust gene silencing in pancreatic macrophages in vivo.

Nucleic acid therapeutics have been utilized for gene regulation, and their recent advancement has led to approval of novel drugs for liver-related disorders. However, systemic extrahepatic delivery remains challenging. Here, we report newly designed mannose-conjugated oligonucleotides for delivering oligonucleotides to macrophages by leveraging the mannose receptor, C-type 1 (MRC1, CD206), which is abundantly expressed in macrophages. We investigated the relationship between cellular uptake and multivalency (mono to tetra) of mannose ligands or linker length and selected a trivalent-mannose ligand. Trivalent-mannose (Man3)-conjugated siRNA induced concentration-dependent gene silencing in both human CD206-overexpressing cells and human macrophages in vitro. After subcutaneous injection into mice, we observed a high distribution of Man3-conjugated oligonucleotides in the liver and pancreata as well as cellular uptake into Kupffer cells and pancreatic macrophages. A single subcutaneous injection of Man3-conjugated siRNA (10 mg/kg) targeting ß2-microglobulin (B2M) silenced B2m mRNA expression by ∼50% and decreased its protein levels in mouse pancreatic macrophages compared to those in saline-treated mice. Of note, multiple subcutaneous injections decreased B2m gene expression and B2M protein levels by ∼80% and ∼85%, respectively. These results show that mannose-conjugation with oligonucleotides is expected to help deliver oligonucleotides to macrophages and regulate gene expression in vivo, particularly in the pancreas.

Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation.

Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.

Deficiency of the kidney tubular angiotensin II type1 receptor-associated protein ATRAP exacerbates streptozotocin-induced diabetic glomerular injury via reducing protective macrophage polarization.

Although activation of the renin-angiotensin system and of its glomerular components is implicated in the pathogenesis of diabetic nephropathy, the functional roles of the tubular renin-angiotensin system with AT1 receptor signaling in diabetic nephropathy are unclear. Tissue hyperactivity of the renin-angiotensin system is inhibited by the angiotensin II type 1 receptor-associated protein ATRAP, which negatively regulates receptor signaling. The highest expression of endogenous ATRAP occurs in the kidney, where it is mainly expressed by tubules but rarely in glomeruli. Here, we found that hyperactivation of angiotensin II type 1 receptor signaling in kidney tubules exacerbated diabetic glomerular injury in a mouse model of streptozotocin-induced diabetic nephropathy. These phenomena were accompanied by decreased expression of CD206, a marker of alternatively activated and tissue-reparative M2 macrophages, in the kidney tubulointerstitium. Additionally, adoptive transfer of M2- polarized macrophages into diabetic ATRAP-knockout mice ameliorated the glomerular injury. As a possible mechanism, the glomerular mRNA levels of tumor necrosis factor-α and oxidative stress components were increased in diabetic knockout mice compared to non-diabetic knockout mice, but these increases were ameliorated by adoptive transfer. Furthermore, proximal tubule-specific ATRAP downregulation reduced tubulointerstitial expression of CD206, the marker of M2 macrophages in diabetic mice. Thus, our findings indicate that tubular ATRAP-mediated functional modulation of angiotensin II type 1 receptor signaling modulates the accumulation of tubulointerstitial M2 macrophages, thus affecting glomerular manifestations of diabetic nephropathy via tubule-glomerular crosstalk.

ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration.

In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5-fluorouracil-induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis-regulatory elements in progenitor cell-related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY-dependent histone acetylation promoted differentiation into CD48+ progenitor cells. Collectively, these results indicate that the dynamic control of ACLY-dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.

Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.

Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated T cells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.

Immunoregulatory Monocyte Subset Promotes Metastasis Associated With Therapeutic Intervention for Primary Tumor.

Systemic and local inflammation associated with therapeutic intervention of primary tumor occasionally promotes metastatic recurrence in mouse and human. However, it remains unclear what types of immune cells are involved in this process. Here, we found that the tissue-repair-promoting Ym1+Ly6Chi monocyte subset expanded as a result of systemic and local inflammation induced by intravenous injection of lipopolysaccharide or resection of primary tumor and promoted lung metastasis originating from circulating tumor cells (CTCs). Deletion of this subset suppressed metastasis induced by the inflammation. Furthermore, transfer of Ym1+Ly6Chi monocytes into naïve mice promoted lung metastasis in the mice. Ym1+Ly6Chi monocytes highly expressed matrix metalloproteinase-9 (MMP-9) and CXCR4. MMP-9 inhibitor and CXCR4 antagonist decreased Ym1+Ly6Chi-monocyte-promoted lung metastasis. These findings indicate that Ym1+Ly6Chi monocytes are therapeutic target cells for metastasis originating from CTCs associated with systemic and local inflammation. In addition, these findings provide a novel predictive cellular biomarker for metastatic recurrence after intervention for primary tumor.

Precision modeling of gall bladder cancer patients in mice based on orthotopic implantation of organoid-derived tumor buds.

Genetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

Downregulated IRF8 in Monocytes and Macrophages of Patients with Systemic Sclerosis May Aggravate the Fibrotic Phenotype.

Monocytes and macrophages may be involved in the pathogenesis of systemic sclerosis (SSc); however, the etiology and regulation of monocyte and macrophage function in SSc remain unknown. IRF8 is a transcriptional regulator that is essential for the differentiation and function of monocytes and macrophages and thus may be involved in the regulation of macrophage phenotypes in SSc fibrosis. In this study, we measured IRF8 levels in circulating monocytes of 26 patients with SSc (diffuse cutaneous SSc, n = 11; limited cutaneous SSc, n = 15) and 14 healthy controls. IRF8 levels were significantly suppressed in monocytes of patients with diffuse cutaneous SSc and correlated negatively with the modified Rodnan total skin thickness score. Next, we assessed expression levels of cell surface markers, cytokine profiles, and components of extracellular matrix in IRF8-silenced monocyte-derived macrophages. IRF8-silenced monocyte-derived macrophages displayed an M2 phenotype and significantly upregulated mRNA and protein levels of profibrotic factors and extracellular matrix components. Finally, we assessed skin fibrosis in myeloid cell-specific IRF8 conditional knockout (Irf8flox/flox; Lyz2Cre/+) mice and found upregulated mRNA levels of extracellular matrix components and increased bleomycin-induced skin fibrosis. In conclusion, altered IRF8 regulation in monocytes and macrophages may be involved in SSc pathogenesis.

A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

Reduction of gender-associated M2-like tumor-associated macrophages in the tumor microenvironment of patients with pancreatic cancer after neoadjuvant chemoradiotherapy.

PURPOSE: This study aimed to investigate gender-dependent antitumor immune response to neoadjuvant chemoradiotherapy (NACRT) in pancreatic ductal adenocarcinoma (PDAC) patients. METHODS: This study enrolled 58 patients (25 females and 33 males) with borderline resectable PDAC who underwent R0 surgical resection after NACRT. The resected tumor specimens were analyzed for tumor-associated macrophages (TAMs); tumor-infiltrating lymphocytes (CD8+ and CD4+ T cells); regulatory T cells; and IRF-5-expressing cells using immunohistochemical staining for CD163, CD204, CD8, CD4, Foxp3, and IRF-5 antigen. The relationship between clinicopathological features and clinical outcomes was evaluated using multivariate Cox proportional hazard analysis. RESULTS: Females had longer overall survival (P = .044) and relapse-free survival (P = .044) than males. The CD204+ TAM number was significantly lower in females than in males (P = .009). No significant difference occurred between female and male patients in other tumor-infiltrating immune cells. IRF-5+ cell number was significantly higher in female patients (P = .002). Negative correlation occurred between CD204+ cells and IRF-5-positive cells (P = .003, r = -.385). CONCLUSIONS: Female gender was an independent prognostic factor possibly due to the greater reduction in CD204+ TAM infiltration in tumors after NACRT. The beneficial effects of NACRT on TAMs' infiltration might be associated with gender-dependent IRF-5 expression.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

Epigenetic and transcriptional regulation of osteoclast differentiation.

Osteoclasts are derived from mononuclear phagocyte lineage cells and are indispensable for bone resorption. Recent findings suggest that fetal yolk sac macrophage progenitors give rise to neonatal osteoclasts, while hematopoietic stem cell-derived cells, such as monocytes, contribute to maintaining osteoclast syncytia in vivo. Osteoclast differentiation is dependent on macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) signaling that mediates global epigenetic and transcriptional changes. PU.1 is a transcription factor that establishes cell type-specific enhancer landscapes in osteoclast precursors and mature osteoclasts by collaborating with interferon regulatory factor-8 (IRF8) and nuclear factor of activated T-cells (NFATc1), respectively. Irf8 and Nfatc1 genes are tightly controlled by epigenetic mechanisms such as DNA methylation and histone modifications during osteoclastogenesis. Thus, key transcription factors orchestrate osteoclast-specific transcription regulatory networks through epigenetic modifications. In this review, we discuss recent advances in our understanding of the molecular mechanisms involved in osteoclast development.

Excessive EP4 Signaling in Smooth Muscle Cells Induces Abdominal Aortic Aneurysm by Amplifying Inflammation.

OBJECTIVE: Excessive prostaglandin E2 production is a hallmark of abdominal aortic aneurysm (AAA). Enhanced expression of prostaglandin E2 receptor EP4 (prostaglandin E receptor 4) in vascular smooth muscle cells (VSMCs) has been demonstrated in human AAAs. Although moderate expression of EP4 contributes to vascular homeostasis, the roles of excessive EP4 in vascular pathology remain uncertain. We aimed to investigate whether EP4 overexpression in VSMCs exacerbates AAAs. Approach and Results: We constructed mice with EP4 overexpressed selectively in VSMCs under an SM22α promoter (EP4-Tg). Most EP4-Tg mice died within 2 weeks of Ang II (angiotensin II) infusion due to AAA, while nontransgenic mice given Ang II displayed no overt phenotype. EP4-Tg developed much larger AAAs than nontransgenic mice after periaortic CaCl2 application. In contrast, EP4fl/+;SM22-Cre;ApoE-/- and EP4fl/+;SM22-Cre mice, which are EP4 heterozygous knockout in VSMCs, rarely exhibited AAA after Ang II or CaCl2 treatment, respectively. In Ang II-infused EP4-Tg aorta, Ly6Chi inflammatory monocyte/macrophage infiltration and MMP-9 (matrix metalloprotease-9) activation were enhanced. An unbiased analysis revealed that EP4 stimulation positively regulated the genes binding cytokine receptors in VSMCs, in which IL (interleukin)-6 was the most strongly upregulated. In VSMCs of EP4-Tg and human AAAs, EP4 stimulation caused marked IL-6 production via TAK1 (transforming growth factor-ß-activated kinase 1), NF-κB (nuclear factor-kappa B), JNK (c-Jun N-terminal kinase), and p38. Inhibition of IL-6 prevented Ang II-induced AAA formation in EP4-Tg. In addition, EP4 stimulation decreased elastin/collagen cross-linking protein LOX (lysyl oxidase) in both human and mouse VSMCs. CONCLUSIONS: Dysregulated EP4 overexpression in VSMCs promotes inflammatory monocyte/macrophage infiltration and attenuates elastin/collagen fiber formation, leading to AAA exacerbation.

[Progress in single-cell analysis of hematopoiesis].

The mechanism underlying production of various types of blood cells from hematopoietic stem and progenitor cells has been a central theme in hematology. Conventionally, hematopoietic cell populations are analyzed by cell surface markers to judge cell types and differentiation stages, and by transplantation assays to assess differentiation potential. Recently, however, next-generation sequencing technology has enabled single-cell transcriptome and epigenome analyses and cell barcoding-based lineage tracing during unperturbed hematopoiesis. These innovative assays revealed that each cell population is extensively heterogenous. Many cells within hematopoietic stem cell populations may not be multipotent, and conversely, hematopoietic progenitor cells often display self-renewal capacity. Moreover, cells tend to make their lineage choice much earlier than previously thought. Altogether, these results challenge the current hierarchical differentiation models and propose new continuous models. Single-cell analyses are expected to greatly contribute to our understanding of normal and abnormal hematopoiesis and to the development of new therapies for blood disorders.

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice.

Dendritic cells (DCs), which are vital for immune responses, are derived from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). DC lineage fate decisions occurring at stages much earlier than CDPs have recently been recognized, yet the mechanism remains elusive. By single-cell RNA-sequencing, in vivo cell transfer experiments, and an assay for transposase-accessible chromatin sequencing using wild-type, IRF8-GFP chimera knock-in or IRF8-knockout mice, we demonstrate that IRF8 regulates chromatin at the lymphoid-primed multipotent progenitor (LMPP) stage to induce early commitment toward DCs. A low but significant expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. These IRF8+ LMPPs were derived from IRF8- LMPPs and predominantly produced DCs, especially classical DC1s, potentially via known progenitors, such as monocyte-DC progenitors, CDPs, and preclassical DCs. IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils, or lymphocytes. Although IRF8- and IRF8+ LMPPs displayed very similar global gene expression patterns, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8- LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, biasing their fate decision toward DCs.