AML/Normal Progenitor Balance Instead of Total Tumor Load (MRD) Accounts for Prognostic Impact of Flowcytometric Residual Disease in AML

Measurable residual disease (MRD) in AML, assessed by multicolor flow cytometry, is an important prognostic factor. Progenitors are key populations in defining MRD, and cases of MRD involving these progenitors are calculated as percentage of WBC and referred to as white blood cell MRD (WBC-MRD). Two main compartments of WBC-MRD can be defined: (1) the AML part of the total primitive/progenitor (CD34+, CD117+, CD133+) compartment (referred to as primitive marker MRD; PM-MRD) and (2) the total progenitor compartment (% of WBC, referred to as PM%), which is the main quantitative determinant of WBC-MRD. Both are related as follows: WBC-MRD = PM-MRD × PM%. We explored the relative contribution of each parameter to the prognostic impact. In the HOVON/SAKK study H102 (300 patients), based on two objectively assessed cut-off points (2.34% and 10%), PM-MRD was found to offer an independent prognostic parameter that was able to identify three patient groups with different prognoses with larger discriminative power than WBC-MRD. In line with this, the PM% parameter itself showed no prognostic impact, implying that the prognostic impact of WBC-MRD results from the PM-MRD parameter it contains. Moreover, the presence of the PM% parameter in WBC-MRD may cause WBC-MRD false positivity and WBC-MRD false negativity. For the latter, at present, it is clinically relevant that PM-MRD ≥ 10% identifies a patient sub-group with a poor prognosis that is currently classified as good prognosis MRDnegative using the European LeukemiaNet 0.1% consensus MRD cut-off value. These observations suggest that residual disease analysis using PM-MRD should be conducted.

Dynamic association of IκBα to chromatin is regulated by acetylation and cleavage of histone H4

ABSTRACTIκBs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for IκB homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied.We here show that serine 32-36 phosphorylation of IκBα favors its binding with nucleosomes and demonstrated that p-IκBα association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-IκBα binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-IκBα chromatin binding and impaired goblet cell differentiation, comparable to IκBα deletion. Together our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-IκBα at specific stem cell compartments.

A biomechanical switch regulates the transition towards homeostasis in esophageal epithelium

Epithelial cells are highly dynamic and can rapidly adapt their behavior in response to tissue perturbations and increasing tissue demands. However, the processes that finely control these responses and, particularly, the mechanisms that ensure the correct switch to and from normal tissue homeostasis are largely unknown. Here we explore changes in cell behavior happening at the interface between postnatal development and homeostasis in the epithelium of the mouse esophagus, as a physiological model exemplifying a rapid but controlled tissue growth transition. Single cell RNA sequencing and histological analysis of the mouse esophagus reveal significant mechanical changes in the epithelium upon tissue maturation. Organ stretching experiments further indicate that tissue strain caused by the differential growth of the mouse esophagus relative to the entire body promotes the emergence of a defined committed population in the progenitor compartment as homeostasis is established. Our results point to a simple mechanism whereby the mechanical changes experienced at the whole tissue level are integrated with those “sensed” at the cellular level to control epithelial cell behavior and tissue maintenance.

Transplacental Innate Immune Training via Maternal Microbial Exposure: Role of XBP1-ERN1 Axis in Dendritic Cell Precursor Programming

We recently reported that offspring of mice treated during pregnancy with the microbial-derived immunomodulator OM-85 manifest striking resistance to allergic airways inflammation, and localized the potential treatment target to fetal conventional dendritic cell (cDC) progenitors. Here, we profile maternal OM-85 treatment-associated transcriptomic signatures in fetal bone marrow, and identify a series of immunometabolic pathways which provide essential metabolites for accelerated myelopoiesis. Additionally, the cDC progenitor compartment displayed treatment-associated activation of the XBP1-ERN1 signalling axis which has been shown to be crucial for tissue survival of cDC, particularly within the lungs. Our forerunner studies indicate uniquely rapid turnover of airway mucosal cDCs at baseline, with further large-scale upregulation of population dynamics during aeroallergen and/or pathogen challenge. We suggest that enhanced capacity for XBP1-ERN1-dependent cDC survival within the airway mucosal tissue microenvironment may be a crucial element of OM-85-mediated transplacental innate immune training which results in postnatal resistance to airway inflammatory disease.

Quantitative lineage analysis identifies a long-term progenitor niche for the hepato-pancreato-biliary organ system

SummarySingle cell-based studies have revealed tremendous cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degree of plasticity during organogenesis1–5.The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including liver, pancreas, gallbladder, and extra-hepatic bile ducts6, 7. Experimental manipulation of various developmental signals in the mouse embryo underscored an important cellular plasticity in this embryonic territory6, 8. This is also reflected in the existence of human genetic syndromes as well as congenital or environmentally-caused human malformations featuring multiorgan phenotypes in liver, pancreas and gallbladder6, 8. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary, and pancreatic structures are not yet established. Here, we combine computational modelling approaches with genetic lineage tracing to assess the tissue dynamics accompanying the ontogeny of the hepato-pancreato-biliary organ system. We show that a long-term multipotent progenitor domain persists at the border between liver and pancreas, even after pancreatic fate is specified, contributing to the formation of several organ derivatives, including the liver. Moreover, using single-cell RNA sequencing we define a specialized niche that possibly supports such long-term cell fate plasticity.

Transplacental innate immune training via maternal microbial exposure: the XBP1-ERN1 axis in programming dendritic cell precursors

We recently reported that the offspring of mice treated during pregnancy with the microbial-derived immunomodulator OM-85 manifest striking resistance postnatally to allergic airways inflammation, and localised the potential treatment target to the fetal cDC progenitor compartment which expands to increase the pool of precursors available at birth, enabling accelerated postnatal seeding of the lung mucosal cDC network required for establishment of immunological homeostasis in the airways. Here, we profile maternal OM-85 treatment-associated transcriptomic signatures in fetal bone marrow, and identify a series of immunometabolic pathways which provide essential metabolites for accelerated myelopoiesis, that are hallmarks of classical “immune training”. In addition, the cDC progenitor compartment displayed treatment-associated activation of the XBP1-ERN1 signalling axis which has previously been shown to be essential for tissue survival of cDC, particularly within the lung microenvironment. Our forerunner studies indicate uniquely rapid turnover of airway mucosal cDCs at baseline, with further large-scale upregulation of population dynamics during aeroallergen and/or pathogen challenge. XBP1-ERN1 signalling plays a key role in mitigation of ER stress-associated toxicity which frequently accompanies DC hyper-activation during intense immunoinflammatory responses, and we suggest that enhanced capacity for XBP1-ERN1-dependent cDC survival within the airway mucosal tissue microenvironment may be a crucial element of the OM-85-mediated transplacental “innate immune training” process which results in enhanced resistance to airway inflammatory disease during the high-risk early postnatal period.

Modeling the Behavior of Hematopoietic Compartments from Stem to Red Cells in Murine Steady State and Stress Hematopoiesis

Maintenance of the blood cell system throughout the lifetime of an organism at steady state and in response to stress requires longevity and integrity of the hematopoietic stem cell pool (HSC). Modeling of erythropoiesis has been previously proposed by solely considering late stages of erythropoiesis, but a model based on all hematopoietic stem and progenitor compartments, efficient in steady state as well as in stress hematopoiesis, has never been proposed. Here we report an erythropoiesis model based on a steady state approach, further complexified by integrating regulatory processes in order to establish a model able to recapitulate steady state and acute stress erythropoiesis. As a first step, we defined a stochastic model (without regulation) relied upon a minimum of 6 cell-amplification compartments to ensure a steady state production of erythroid cells based on: i- the number of LT-HSC, ii- the number of terminal mitosis to enable red cell production from mature erythroid progenitor, iii-the differential probability of differentiation versus self-renewal in each progenitor compartment, and the division rates from iv- LT-HSC and v-last mature erythroid progenitor compartment. Computer analysis was performed using Python language with an optimization method (CMA-ES: Covariance Matric Adaptation- Evolution Strategy). This model mimics well what is observed in vivo with the LT-HSC, ST-HSC, MPP, CMP, MEP and mature red blood cell compartments. We next assessed the effects of an acute stress targeting mature red blood cells such as phenylhydrazine treatment (PHZ) on these different progenitor compartments, from LT-HSC to mature red blood cells. PHZ was i.p administered (one dose of 60 mg/ Kg) , and mice were analyzed at days 0,1,3, 5, 10, 16, and 28 for blood parameters, for the proportion of the different bone marrow (BM) and spleen progenitor compartments as well as their percentages into cell cycle (BrdU incorporation), and apoptosis (Annexin V labelling). PHZ treatment induced a severe anemia, characterized by a strong fall of the hematocrit (30-50%) at day 3, followed by a rapid 10-day recovery. Regarding BM progenitor compartments, our results showed that, together with a stability in the number of BM cells, PHZ treatment drastically reduced at day 3-5 all progenitor compartments with a direct flush from LT-HSC (-75%) to mature cells without modifying apoptosis, proliferation or egress of progenitors from BM to compensate the loss of mature cells. In a second phase, a gradual replenishment of all progenitor compartments, from the most mature (MEP, CMP) to intermediate (MPP) and immature (ST-HSC and LT-HSC), with oscillations around the equilibrium reached at day 28, was observed. Such recovery was accompanied by a recruitment of LT and ST-HSC into cell cycle: ¼ of all LT-HSC and ST-HSC cycling at day 10 compared to 2% in steady state. The former mathematical model was then applied to PHZ treatment, but a strong discrepancy occurred in the time course of red blood cell recovery, which was 3 times longer than what was observed in vivo (30 days instead of 10 days). We therefore added to the stochastic model regulations between the different compartments based on what is observed with cytokines in vivo and the modifications of the size of the progenitor compartments. This mathematic model, now complemented by addition of these regulations, recapitulated the evolution of the different progenitor compartments observed after PHZ treatment as well as in steady state. To our knowledge, this is the first multi-step modeling of hematopoiesis able to recapitulate steady state as well as stressed hematopoiesis, by taking into account the current progenitor compartments of the BM, and able to explain at the single cell level what happens during hematopoietic differentiation process. This is also the first model demonstrating that hematopoietic regulation just like cytokines effects is necessary during stress hematopoiesis but dispensable during steady state hematopoiesis. Modeling of hematopoiesis integrating heterogeneity of the cell populations as described here will allow us to study the effects of abnormal event integration in a cell population just like the effects of the emergence of a single cancer-initiating cell. Disclosures No relevant conflicts of interest to declare.