Integrin-α9 overexpression underlies the niche-independent maintenance of leukemia stem cells in acute myeloid leukemia.

Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML.

Flt3- and Tie2-Cre tracing identifies regeneration in sepsis from multipotent progenitors but not hematopoietic stem cells.

In response to infections and stress, hematopoiesis rapidly enhances blood and immune cell production. The stage within the hematopoietic hierarchy that accounts for this regeneration is unclear under natural conditions in vivo. We analyzed by differentiation tracing, using inducible Tie2- or Flt3-driven Cre recombinase, the roles of mouse hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). During polymicrobial sepsis, HSCs responded transcriptionally and increased their proliferation and cell death, yet HSC differentiation rates remained at steady-state levels. HSC differentiation was also independent from the ablation of various cellular compartments-bleeding, the antibody-mediated ablation of granulocytes or B lymphocytes, and genetic lymphocyte deficiency. By marked contrast, the fate mapping of MPPs in polymicrobial sepsis identified these cells as a major source for accelerated myeloid cell production. The regulation of blood and immune cell homeostasis by progenitors rather than stem cells may ensure a rapid response while preserving the integrity of the HSC population.

Immune competence and spleen size scale with colony status in the naked mole-rat.

Naked mole-rats (NM-R; Heterocephalus glaber) live in multi-generational colonies with a social hierarchy, and show low cancer incidence and long life-spans. Here we asked if an immune component might underlie such extreme physiology. The largest lymphoid organ is the spleen, which plays an essential role in responding to immunological insults and may participate in combating cancer and slowing ageing. We investigated the anatomy, molecular composition and function of the NM-R spleen using RNA-sequencing and histological analysis in healthy NM-Rs. Spleen size in healthy NM-Rs showed considerable inter-individual variability, with some animals displaying enlarged spleens. In all healthy NM-Rs, the spleen is a major site of adult haematopoiesis under normal physiological conditions. However, myeloid-to-lymphoid cell ratio is increased and splenic marginal zone showed markedly altered morphology when compared to other rodents. Healthy NM-Rs with enlarged spleens showed potentially better anti-microbial profiles and were much more likely to have a high rank within the colony. We propose that the anatomical plasticity of the spleen might be regulated by social interaction and gives immunological advantage to increase the lifespan of higher-ranked animals.

Transmission of trained immunity and heterologous resistance to infections across generations.

Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.

Correction: CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BCG vaccination in humans inhibits systemic inflammation in a sex-dependent manner.

BACKGROUNDInduction of innate immune memory, also termed trained immunity, by the antituberculosis vaccine bacillus Calmette-Guérin (BCG) contributes to protection against heterologous infections. However, the overall impact of BCG vaccination on the inflammatory status of an individual is not known; while induction of trained immunity may suggest increased inflammation, BCG vaccination has been epidemiologically associated with a reduced incidence of inflammatory and allergic diseases.METHODSWe investigated the impact of BCG (BCG-Bulgaria, InterVax) vaccination on systemic inflammation in a cohort of 303 healthy volunteers, as well as the effect of the inflammatory status on the response to vaccination. A targeted proteome platform was used to measure circulating inflammatory proteins before and after BCG vaccination, while ex vivo Mycobacterium tuberculosis- and Staphylococcus aureus-induced cytokine responses in peripheral blood mononuclear cells were used to assess trained immunity.RESULTSWhile BCG vaccination enhanced cytokine responses to restimulation, it reduced systemic inflammation. This effect was validated in 3 smaller cohorts, and was much stronger in men than in women. In addition, baseline circulating inflammatory markers were associated with ex vivo cytokine responses (trained immunity) after BCG vaccination.CONCLUSIONThe capacity of BCG to enhance microbial responsiveness while dampening systemic inflammation should be further explored for potential therapeutic applications.FUNDINGNetherlands Organization for Scientific Research, European Research Council, and the Danish National Research Foundation.

BCG Vaccination in Humans Elicits Trained Immunity via the Hematopoietic Progenitor Compartment.

Induction of trained immunity by Bacille-Calmette-Guérin (BCG) vaccination mediates beneficial heterologous effects, but the mechanisms underlying its persistence and magnitude remain elusive. In this study, we show that BCG vaccination in healthy human volunteers induces a persistent transcriptional program connected to myeloid cell development and function within the hematopoietic stem and progenitor cell (HSPC) compartment in the bone marrow. We identify hepatic nuclear factor (HNF) family members 1a and b as crucial regulators of this transcriptional shift. These findings are corroborated by higher granulocyte numbers in BCG-vaccinated infants, HNF1 SNP variants that correlate with trained immunity, and elevated serum concentrations of the HNF1 target alpha-1 antitrypsin. Additionally, transcriptomic HSPC remodeling was epigenetically conveyed to peripheral CD14+ monocytes, displaying an activated transcriptional signature three months after BCG vaccination. Taken together, transcriptomic, epigenomic, and functional reprogramming of HSPCs and peripheral monocytes is a hallmark of BCG-induced trained immunity in humans.

ß-Glucan-Induced Trained Immunity Protects against Leishmania braziliensis Infection: a Crucial Role for IL-32.

American tegumentary leishmaniasis is a vector-borne parasitic disease caused by Leishmania protozoans. Innate immune cells undergo long-term functional reprogramming in response to infection or Bacillus Calmette-Guérin (BCG) vaccination via a process called trained immunity, conferring non-specific protection from secondary infections. Here, we demonstrate that monocytes trained with the fungal cell wall component ß-glucan confer enhanced protection against infections caused by Leishmania braziliensis through the enhanced production of proinflammatory cytokines. Mechanistically, this augmented immunological response is dependent on increased expression of interleukin 32 (IL-32). Studies performed using a humanized IL-32 transgenic mouse highlight the clinical implications of these findings in vivo. This study represents a definitive characterization of the role of IL-32γ in the trained phenotype induced by ß-glucan or BCG, the results of which improve our understanding of the molecular mechanisms governing trained immunity and Leishmania infection control.

Analysis of High-Dimensional Phenotype Data Generated by Mass Cytometry or High-Dimensional Flow Cytometry.

Recent advances in single cell multi-omics methodologies significantly expand our understanding of cellular heterogeneity, particularly in the field of immunology. Today's state-of-the-art flow and mass cytometers can detect up to 50 parameters to comprehensively characterize the identity and function of individual cells within a heterogeneous population. As a consequence, the increasing number of parameters that can be detected simultaneously also introduces substantial complexity for the experimental setup and downstream data processing. However, this challenge in data analysis fostered the development of novel bioinformatic tools to fully exploit the high-dimensional data. These tools will eventually replace cumbersome serial, manual gating in the two-dimensional space driven by a priori knowledge, which still represents the gold standard in flow cytometric analysis, to meet the needs of the today's immunologist. To this end, we provide guidelines for a high-dimensional cytometry workflow including experimental setup, panel design, fluorescent spillover compensation, and data analysis.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

C/EBP-Induced Transdifferentiation Reveals Granulocyte-Macrophage Precursor-like Plasticity of B Cells.

The lymphoid-myeloid transdifferentiation potentials of members of the C/EBP family (C/EBPα, ß, δ, and ε) were compared in v-Abl-immortalized primary B cells. Conversion of B cells to macrophages was readily induced by the ectopic expression of any C/EBP, and enhanced by endogenous C/EBPα and ß activation. High transgene expression of C/EBPß or C/EBPε, but not of C/EBPα or C/EBPδ, also induced the formation of granulocytes. Granulocytes and macrophages emerged in a mutually exclusive manner. C/EBPß-expressing B cells produced granulocyte-macrophage progenitor (GMP)-like progenitors when subjected to selective pressure to eliminate lymphoid cells. The GMP-like progenitors remained self-renewing and cytokine-independent, and continuously produced macrophages and granulocytes. In addition to their suitability to study myelomonocytic lineage bifurcation, lineage-switched GMP-like progenitors could reflect the features of the lympho-myeloid lineage switch observed in leukemic progression.

A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells.

Runx1 is essential for the generation of hematopoietic stem cells (HSCs) and is frequently mutated in human leukemias. However, the cis-regulatory mechanisms modulating the Runx1 gene expression remain to be elucidated. Herewith, we report the identification of an intronic Runx1 enhancer, Runx1 +24 mouse conserved noncoding element (mCNE), using a combinatorial in silico approach involving comparative genomics and retroviral integration sites mapping. The Runx1 +24 mCNE was found to possess hematopoietic-specific enhancer activity in both zebrafish and mouse models. Significantly, this enhancer is active specifically in hemogenic endothelial cells (ECs) at sites where the de novo generation of HSCs occurs. The activity of this enhancer is also strictly restricted to HSCs within the hematopoietic compartment of the adult bone marrow. We anticipate that Runx1 +24 mCNE HSC enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic ECs/HSCs behavior in vivo, and consequently become an invaluable tool for research on stem cell and cancer biology.