Blood Cell Homeostasis Research Articles

#2505 PTEN ablation in osteocytes exacerbates anemia in mice with subtotal nephrectomy

Abstract Background and Aims The identification of novel roles for bone as a principal endocrine organ has grown in recent decades, being now recognized for its contribution to the regulation of numerous systemic functions. In previous studies, we observed that 5/6Nx mice with osteocytic deletion of phosphatase and tensin homolog (PTEN), exhibited higher bone volume in 5/6Nx mice, consistent with studies under normal renal conditions. However, we also noted a decrease in blood hemoglobin levels, prompting further exploration. This study aims to elucidate the causative actions by which specific PTEN deletion in osteocytes influences red blood cell (RBC) homeostasis and to identify its possible involvement in anemia development. Method To achieve this goal, we used mice with targeted PTEN deletion in osteocytes (PTEN Ocy-cKO) by crossing DMP1-Cre ± and PTENflox/flox mice, subjected to subtotal nephrectomy (5/6Nx) at 2 months of age. Two weeks post-surgery, bone and blood samples were collected for plasma biochemistry, blood count, RBC membrane integrity, and preliminary bone+bone marrow proteomic analysis. Cre-negative mice and animals with normal renal function were included as controls. Results Our findings revealed that PTEN Ocy-cKO mice, under both normal and reduced renal function conditions, showed higher bone volume than corresponding controls. In terms of blood homeostasis, PTEN Ocy-cKO mice with normal renal function showed splenomegaly and a lower number of circulating RBCs. Notably, PTEN Ocy-cKO mice undergoing 5/6Nx experienced a profound decrease in RBC number in only two weeks, independent of renal erythropoietin expression. Further exploration ruled out bone marrow failure as a cause, as the erythroid progenitor (CD45-CD71+Ter119+) population remained similar in both groups. Extramedullary hematopoiesis was observed in the spleen, in addition to increased circulating reticulocyte number in the PTEN Ocy-cKO mice. Finally, altered RBC membrane protein patterns in PTEN Ocy-cKO mice suggested reduced mechanical/osmotic resistance. Preliminary proteomic analysis identified downregulation of Slc4a1 (Band3) and Phosphofructokinase (Pfkm), whose deficiencies have been previously linked to anemia. Conclusion In conclusion, our results propose that osteocytes contribute to RBC homeostasis through a pathway downstream of PTEN, and open a new avenue for understanding anemia in chronic kidney disease and a potential target for future treatments.

Nuclear receptor Rev-erbα role in fine-tuning erythropoietin gene expression

AbstractThe regulation of red blood cell (RBC) homeostasis by erythropoietin (EPO) is critical for O2 transport and maintaining the adequate number of RBCs in vertebrates. Therefore, dysregulation in EPO synthesis results in disease conditions such as polycythemia in the case of excessive EPO production and anemia, which occurs when EPO is inadequately produced. EPO plays a crucial role in treating anemic patients; however, its overproduction can increase blood viscosity, potentially leading to fatal heart failure. Consequently, the identification of druggable transcription factors and their associated ligands capable of regulating EPO offers a promising therapeutic approach to address EPO-related disorders. This study unveils a novel regulatory mechanism involving 2 pivotal nuclear receptors (NRs), Rev-ERBA (Rev-erbα, is a truncation of reverse c-erbAa) and RAR-related orphan receptor A (RORα), in the control of EPO gene expression. Rev-erbα acts as a cell-intrinsic negative regulator, playing a vital role in maintaining erythropoiesis at the correct level. It accomplishes this by directly binding to newly identified response elements within the human and mouse EPO gene promoter, thereby repressing EPO production. These findings are further supported by the discovery that a Rev-erbα agonist (SR9011) effectively suppresses hypoxia-induced EPO expression in mice. In contrast, RORα functions as a positive regulator of EPO gene expression, also binding to the same response elements in the promoter to induce EPO production. Finally, the results of this study revealed that the 2 NRs, Rev-erbα and RORα, influence EPO synthesis in a negative and positive manner, respectively, suggesting that the modulating activity of these 2 NRs could provide a method to target disorders linked with EPO dysregulation.

Numerous Serine/Threonine Kinases Affect Blood Cell Homeostasis in Drosophila melanogaster.

Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.

PIEZO Ion Channels in Cardiovascular Functions and Diseases.

The cardiovascular system provides blood supply throughout the body and as such is perpetually applying mechanical forces to cells and tissues. Thus, this system is primed with mechanosensory structures that respond and adapt to changes in mechanical stimuli. Since their discovery in 2010, PIEZO ion channels have dominated the field of mechanobiology. These have been proposed as the long-sought-after mechanosensitive excitatory channels involved in touch and proprioception in mammals. However, more and more pieces of evidence point to the importance of PIEZO channels in cardiovascular activities and disease development. PIEZO channel-related cardiac functions include transducing hemodynamic forces in endothelial and vascular cells, red blood cell homeostasis, platelet aggregation, and arterial blood pressure regulation, among others. PIEZO channels contribute to pathological conditions including cardiac hypertrophy and pulmonary hypertension and congenital syndromes such as generalized lymphatic dysplasia and xerocytosis. In this review, we highlight recent advances in understanding the role of PIEZO channels in cardiovascular functions and diseases. Achievements in this quickly expanding field should open a new road for efficient control of PIEZO-related diseases in cardiovascular functions.

A new role for erythropoietin in the homeostasis of red blood cells

The regulation of red blood cell (RBC) homeostasis is widely assumed to rely on the control of cell production by erythropoietin (EPO) and the destruction of cells at a fixed, species-specific age. In this work, we show that such a regulatory mechanism would be a poor homeostatic solution to satisfy the changing needs of the body. Effective homeostatic control would require RBC lifespan to be variable and tightly regulated. We suggest that EPO may control RBC lifespan by determining CD47 expression in newly formed RBCs and SIRP-α expression in sinusoidal macrophages. EPO could also regulate the initiation and intensity of anti-RBC autoimmune responses that curtail RBC lifespan in some circumstances. These mechanisms would continuously modulate the rate of RBC destruction depending on oxygen availability. The control of RBC lifespan by EPO and autoimmunity emerges as a key mechanism in the homeostasis of RBCs.

Multi-omics integrative analysis revealed characteristic changes in blood cell immunity and amino acid metabolism in a silkworm model of hyperproteinemia

Hyperproteinemia is a serious metabolic disease of both humans and animals characterized by an abnormally high plasma protein concentration (HPPC). Although hyperproteinemia can cause an imbalance in blood cell homeostasis, the functional changes to blood cells remain unclear. Here, a HPPC silkworm model was used to assess changes to the chromatin accessibility and transcript levels of genes related to blood cell metabolism and immune function. The results showed that HPPC enhanced phagocytosis of blood cells, increased chromatin accessibility and transcript levels of genes involved in cell phagocytosis, proliferation, stress, and programmed death, while genes associated with aromatic amino acid metabolism, and antibacterial peptide synthesis were inhibited in blood cells. Further analysis of the chromatin accessibility of the promoter region found that the high chromatin accessibility of genes sensitive to HPPC, was related to histone modifications, including tri-methylation of lysine residue 4 of histone H3 and acetylation of lysine residue 27 of histone H3. Changes to the chromatin accessibility and transcript levels of genes related to immune function and amino acid metabolism in the blood cells of the HPPC silkworm model provided useful references for future studies of the mechanisms underlying epigenomic regulation mediated by hyperproteinemia.

A mechanosensitive vascular niche for Drosophila hematopoiesis

Hematopoietic stem and progenitor cells maintain blood cell homeostasis by integrating various cues provided by specialized microenvironments or niches. Biomechanical forces are emerging as key regulators of hematopoiesis. Here, we report that mechanical stimuli provided by blood flow in the vascular niche control Drosophila hematopoiesis. In vascular niche cells, the mechanosensitive channel Piezo transduces mechanical forces through intracellular calcium upregulation, leading to Notch activation and repression of FGF ligand transcription, known to regulate hematopoietic progenitor maintenance. Our results provide insight into how the vascular niche integrates mechanical stimuli to regulate hematopoiesis.

Alterations of Water-Mineral Homeostatic System in Rabbits Under the Action of Stressor Factors

This study aimed to examine the alteration of circadian structures of electrolytes, trace elements, and white blood cell homeostasis under the action of stressors factors. Male rabbits were used in experimental work. Four-hour urine and blood specimens were collected over a span of 48–72 hours. Total Na (sodium), K (potassium), Ca (calcium), Mg (magnesium), Cu (copper), and Zn (zinc) were analyzed on atomic absorption spectrophotometer. Rhythm’s parameters have been estimated by the nonlinear least square method for sinusoidal rhythms and by dispersion analysis for nonsinusoidal rhythms. Intact rabbits’ chronoperiodical systems of water-mineral and white blood cell homeostasis were characterized by circadian structures. Acrophases of indices of water-mineral and white blood cell homeostasis in intact rabbits mostly had individual nature. Temporal structure of water-mineral and white blood cell homeostasis in rabbits under the action of stress were characterized by infradian rhythms or nonperiodical oscillations.

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 invivo. 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.

The circulatory dynamics of human red blood cell homeostasis: Oxy-deoxy and PIEZO1-triggered changes

The vital function of red blood cells (RBCs) is to mediate the transport of oxygen from lungs to tissues and of CO2 from tissues to lungs. The gas exchanges occur during capillary transits within fractions of a second. Each oxygenation-deoxygenation and deoxygenation-reoxygenation transition on hemoglobin triggers sharp changes in RBC pH, leading to downstream changes in ion fluxes, membrane potential, and cell volume. The dynamics of these changes during the variable periods between capillary transits in vivo remains a mystery inaccessible to study by current methodologies, a knowledge gap on a fundamental physiological process that is the focus of the present study. The use of a computational model of human RBC homeostasis of tested accreditation enabled a detailed investigation of the expected RBC changes during intercapillary transits, with results advancing novel insights and predictions. The predicted rates of relative RBC volume change on oxygenation-deoxygenation (oxy-deoxy) and deoxygenation-reoxygenation transitions were about 1.5%/min and −0.9%/min, respectively, far too slow to allow the cells to reach steady states in the intervals between capillary transits. The amplitude of the oxy-deoxy-reoxygenation volume fluctuations varied in proportion with the duration of the intercapillary transit intervals. Upon capillary entry, oxy-deoxy-induced changes occur concurrently with deformation-induced PIEZO1 channel activation, both processes affecting cell pH, membrane potential, and cell volume during intertransit periods. The model showed that the effects were strictly additive as expected from processes operating independently on the cell’s homeostatic fabric. Analysis of the mechanisms behind these predictions revealed, for the first time, the complex interactions between oxy-deoxy and ion transport processes that ensure the long-term homeostatic stability of RBCs for optimal gas transport in physiological conditions and how these may become altered in diseased states. Possible designs of microfluidic devices to test the model predictions are discussed.

Role of Piezo1 channel in cardiovascular diseases and its research progress in traditional Chinese medicine

Piezo1 channel is a mechanosensitive ion channel that opens in response to shear stress, tension, torsion and a series of mechanical stimulation and turns them into biological signals. It plays an important role in vascular development, vasoconstriction and vasodilation, formation of arterial and venous valves, blood pressure regulation and red blood cell homeostasis, and thus is closely related to cardiovascular system. Recent studies have shown that traditional Chinese medicine(TCM) compounds and their active components can affect Ca~(2+) influx and endothelial cell and platelet function by mediating Piezo1 channel, and regulate thrombosis and endothelial homeostasis, thereby treating cardiovascular diseases. This study mainly reviewed the role of Piezo1 channel in cardiovascular diseases and the prevention and treatment of cardiovascular diseases based on Piezo1 channel in TCM, in order to provide effective reference for the further research of Piezo1 channel in the field of TCM.

Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model

Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration (PPC) and is often clinically complicated by malignant diseases or severe infections. At present, however, research on the molecular mechanism underlying high PPC (HPPC) is scant. Here, an animal model of primary hyperproteinemia was constructed in an invertebrate (Bombyx mori) to investigate the effects of HPPC on circulating blood cells. Results showed that HPPC affected blood cell homeostasis, leading to increased reactive oxygen species levels, and induced programmed cell death dependent on the endoplasmic reticulum-calcium ion signaling pathway. HPPC induced the proliferation of blood cells, mainly granulocytes, by activating the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Supplementation with the endocrine hormone active substance 20E significantly reduced the impact of HPPC on blood cell homeostasis. Thus, we identified a novel signaling pathway by which HPPC affects blood cell homeostasis, which differs from hyperglycemia, hyperlipidemia, and hypercholesterolemia. In addition, we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early detection indicator for hyperproteinemia.

Targeting Host Glycolysis as a Strategy for Antimalarial Development.

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

HLF expression defines the human hematopoietic stem cell state

AbstractHematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages after transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. Although several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population and single-cell transcriptomes of unexpanded and ex vivo cultured cord blood–derived hematopoietic stem and progenitor cells as well as peripheral blood, adult bone marrow, and fetal liver. On the basis of these analyses, we propose the master transcription factor HLF (hepatic leukemia factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter. Most importantly, HLF-expressing cells comprise all stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human HSC state and outline a new approach to continuously mark these cells with high fidelity.

Comparative role of hematological indices for the assessment of in-hospital outcome of heart failure patients

Background. The mutual relation between heart failure (HF) and inflammation is reflected in blood cell homeostasis. Neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR) and platelet-lymphocyte ratio (PLR) were linked to HF severity and prognosis. Aims. Our objective was to compare the three ratios for predicting in-hospital outcome of HF patients, in order to establish which is best suited for clinical practice. Methods. Consecutive HF patients admitted to a Cardiology Department from a tertiary hospital were retrospectively evaluated for inclusion. Readmissions and pathologies modifying the hematological indices were excluded. Extended length of hospital stay (LOS) was considered over 7 d. In-hospital all-cause mortality was evaluated. Results: The hematological indices in heart failure (HI-HF) cohort included 1299 patients with a mean age of 72.35 ± 10.45 years, 51.96% women. 2.85% died during hospitalization. 22.17% had extended LOS. In Cox regression for in-hospital mortality alongside parameters from the OPTIMIZE-HF proposed model, all three ratios were independent predictors of mortality. In Cox regression including NT-proBNP, dyspnea at rest, chronic obstructive pulmonary disease (COPD), age and systolic blood pressure, only MLR was an independent predictor of in-hospital mortality (HR 1.68, 95% CI 1.22 − 2.32, p = .002). In multivariable logistic regression, all three ratios independently predicted extended LOS. MLR > 0.48 associated the highest probability (OR 1.76, 95% CI 1.25 − 2.46, p = .001). Conclusions. Hematological indices could be cost-effective and easily available auxiliary biomarkers for in-hospital prognosis of HF patients. We propose MLR > 0.48 as the strongest predictor of in-hospital mortality and prolonged hospitalization.

The vascular niche controls Drosophila hematopoiesis via fibroblast growth factor signaling.

In adult mammals, hematopoiesis, the production of blood cells from hematopoietic stem and progenitor cells (HSPCs), is tightly regulated by extrinsic signals from the microenvironment called 'niche'. Bone marrow HSPCs are heterogeneous and controlled by both endosteal and vascular niches. The Drosophila hematopoietic lymph gland is located along the cardiac tube which corresponds to the vascular system. In the lymph gland, the niche called Posterior Signaling Center controls only a subset of the heterogeneous hematopoietic progenitor population indicating that additional signals are necessary. Here we report that the vascular system acts as a second niche to control lymph gland homeostasis. The FGF ligand Branchless produced by vascular cells activates the FGF pathway in hematopoietic progenitors. By regulating intracellular calcium levels, FGF signaling maintains progenitor pools and prevents blood cell differentiation. This study reveals that two niches contribute to the control ofDrosophila blood cell homeostasis through their differential regulation of progenitors.

Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso

Asymptomatic malaria infections may affect red blood cell (RBC) homeostasis. Reports indicate a role for chronic hemolysis and splenomegaly, however, the underlying processes are incompletely understood. New hematology analysers provide parameters for a more comprehensive analysis of RBC hemostasis. Complete blood counts were analysed in subjects from all age groups (n = 1118) living in a malaria hyperendemic area and cytokines and iron biomarkers were also measured. Subjects were divided into age groups (<2 years, 2-4, 5-14 and ≥15 years old) and clinical categories (smear-negative healthy subjects, asymptomatic malaria and clinical malaria). We found that hemoglobin levels were similar in smear-negative healthy children and asymptomatic malaria children but significantly lower in clinical malaria with a maximum difference of 2.2 g/dl in children <2 years decreasing to 0.1 g/dl in those aged ≥15 years. Delta-He, presenting different hemoglobinization of reticulocytes and RBC, levels were lower in asymptomatic and clinial malaria, indicating a recent effect of malaria on erythropoiesis. Reticulocyte counts and reticulocyte production index (RPI), indicating the erythropoietic capacity of the bone marrow, were higher in young children with malaria compared to smear-negative subjects. A negative correlation between reticulocyte counts and Hb levels was found in asymptomatic malaria (ρ = -0.32, p<0.001) unlike in clinical malaria (ρ = -0.008, p = 0.92). Free-Hb levels, indicating hemolysis, were only higher in clinical malaria. Phagocytozing monocytes, indicating erythophagocytosis, were highest in clinical malaria, followed by asymptomatic malaria and smear-negative subjects. Circulating cytokines and iron biomarkers (hepcidin, ferritin) showed similar patterns. Pro/anti-inflammatory (IL-6/IL-10) ratio was higher in clinical than asymptomatic malaria. Cytokine production capacity of ex-vivo whole blood stimulation with LPS was lower in children with asymptomatic malaria compared to smear-negative healthy children. Bone marrow response can compensate the increased red blood cell loss in asymptomatic malaria, unlike in clinical malaria, possibly because of limited level and length of inflammation. Trial registration: Prospective diagnostic study: ClinicalTrials.gov identifier: NCT02669823. Explorative cross-sectional field study: ClinicalTrials.gov identifier: NCT03176719.

Combating hypoxemia in COVID-19 patients with a natural oxygen carrier, HEMO2Life® (M101)

BackgroundInfection with SARS-CoV-2 is responsible for the COVID-19 crisis affecting the whole world. This virus can provoke acute respiratory distress syndrome (ARDS) leading to overcrowed the intensive care unit (ICU). Over the last months, worldwide experience demonstrated that the ARDS in COVID-19 patients are in many ways “atypical”. The mortality rate in ventilated patients is high despite the application of the gold standard treatment (protective ventilation, curare, prone position, inhaled NO). Several studies suggested that the SARS-CoV-2 could interact negatively on red blood cell homeostasis. Furthermore, SarsCov2 creates Reactive Oxygen Species (ROS), which are toxic and generate endothelial dysfunction. Hypothesis/objective(s)We hypothesis that HEMO2Life® administrated intravenously is safe and could help symptomatically the patient condition. It would increase arterial oxygen content despite lung failure and allow better tissue oxygenation control. The use of HEMO2Life® is also interesting due to its anti-oxidative effect preventing cytokine storm induced by the SARS-CoV-2. Evaluation of the hypothesis: Hemarina is based on the properties of the hemoglobin of the Arenicola marina sea-worm (HEMO2Life®). This extracellular hemoglobin has an oxygen capacity 40 times greater than the hemoglobin of vertebrates. Furthermore, the size of this molecule is 250 times smaller than a human red blood cell, allowing it to diffuse in all areas of the microcirculation, without diffusing outside the vascular sector. It possesses an antioxidative property du a Superoxide Dismutase Activity. This technology has been the subject of numerous publications and HEMO2Life® was found to be well-tolerated and did not induce toxicity. It was administered intravenously to hamsters and rats, and showed no acute effect on heart rate and blood pressure and did not cause microvascular vasoconstriction. In preclinical in vivo models (mice, rats, and dogs), HEMO2Life® has enabled better tissue oxygenation, especially in the brain. This molecule has already been used in humans in organ preservation solutions and the patients showed no abnormal clinical signs. Consequences of the hypothesisThe expected benefits of HEMO2Life® for COVID-19 patients are improved survival, avoidance of tracheal intubation, shorter oxygen supplementation, and the possibility of treating a larger number of patients as molecular respirator without to use an invasive machine.

Abstract 4541: QL315, A CD3 bispecific antibody redirecting T cell activation to the tumor stroma antigen LRRC15

Abstract Leucine-rich repeat containing 15 (LRRC15) antigen is highly expressed on both stromal fibroblasts of multiple solid tumors and on cancer cells of mesenchymal origin. As its expression is very low in normal tissues, targeting LRRC15 has shown therapeutic potential as an antibody-drug conjugate (Demetri, ASCO 2019), which could lead to significant tumor inhibition by solely targeting stromal LRRC15 expression. In this study, we have developed and rank ordered an array of IgG-based CD3 bispecific molecules based on biophysical and biologic attributes and anti-cancer efficiency through T-cell engagement and redirection/activation against LRRC15-expressing tumors. The selected lead, QL315, robustly and conditionally induced T cell activation and proliferation only in the presence of LRRC15-expressing cancer cells with an EC50 in a picomolar range in vitro but not in the coculture with LRRC15-negative cancer cells. Moreover, QL315 strongly induced tumor cell lysis in vitro and led to significant tumor regression and T cell expansion in a mouse xenograft model. Importantly, toxicologic evaluation of QL315 in cynomolgus monkeys showed antibody-like stability and half-life, and good tolerability with no changes of circulating blood cell homeostasis and organ damage. At highest levels, liver enzymes, as well as cytokine release, were transiently elevated but returned to normal within one week of treatment. In conclusion, these data demonstrate drug-like properties, promising anti-tumor capability and good tolerability of the anti-LRRC15/CD3 bispecific antibody, QL315, indicating its potential as a therapeutic option in the treatment of solid tumors of diverse origin by targeting either tumor stromal micro environment or patient cancers expressing LRRC15. Citation Format: Monica Jin, Aaron Kurtzman, Weiwei W. Prior, Allan Chan, Anna McClain, Shenda Gu, Irene Tang, Zijie Cai, Lauren Schwimmer, Hieu Tran, Shihao Chen. QL315, A CD3 bispecific antibody redirecting T cell activation to the tumor stroma antigen LRRC15 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4541.

3019 – CRISPR-BASED ENGINEERING OF A REPORTER ALLELE DEMONSTRATES THAT HLF EXPRESSION DEFINES THE HUMAN HAEMATOPOIETIC STEM CELL STATE

Haematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and are able to regenerate all blood lineages following transplantation. Despite this clear functional definition, the accurate separation of human HSCs from their differentiated progeny can currently only be achieved through combinatorial measurement of multiple surface antigens. By extensive transcriptomic surveying of bulk and single-cell datasets, we have identified the master transcription factor HLF (Hepatic Leukemia Factor) as the most selective and consistently expressed marker gene in HSC-enriched sub-populations of different ontogenetic sources and ex vivo expanded CD34+ cord blood cells. To directly track its expression, we have engineered a genomic reporter strategy that relies on targeting its 3' non-coding region with a Cas9 ribonucleoprotein complex followed by infection with a recombinant AAV6 vector providing a repair template to introduce a fluorescent gene cassette. This approach routinely achieves high reporter insertion efficiency and is capable of selectively labeling the most immature human blood cells on the basis of a single engineered parameter. Most importantly, use of this reporter demonstrates that HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, our results establish HLF as the most defining gene of the human haematopoietic stem cell state and outline a next-generation approach to mark these cells in real-time with unmatched fidelity.