Chronic viral infections persistently alter marrow stroma and impair hematopoietic stem cell fitness.

Chronic viral infections are associated with hematopoietic suppression, bone marrow (BM) failure, and hematopoietic stem cell (HSC) exhaustion. However, how persistent viral challenge and inflammatory responses target BM tissues and perturb hematopoietic competence remains poorly understood. Here, we combine functional analyses with advanced 3D microscopy to demonstrate that chronic infection with lymphocytic choriomeningitis virus leads to (1) long-lasting decimation of the BM stromal network of mesenchymal CXCL12-abundant reticular cells, (2) proinflammatory transcriptional remodeling of remaining components of this key niche subset, and (3) durable functional defects and decreased competitive fitness in HSCs. Mechanistically, BM immunopathology is elicited by virus-specific, activated CD8 T cells, which accumulate in the BM via interferon-dependent mechanisms. Combined antibody-mediated inhibition of type I and II IFN pathways completely preempts degeneration of CARc and protects HSCs from chronic dysfunction. Hence, viral infections and ensuing immune reactions durably impact BM homeostasis by persistently decreasing the competitive fitness of HSCs and disrupting essential stromal-derived, hematopoietic-supporting cues.

Modality Attention and Sampling Enables Deep Learning with Heterogeneous Marker Combinations in Fluorescence Microscopy.

Fluorescence microscopy allows for a detailed inspection of cells, cellular networks, and anatomical landmarks by staining with a variety of carefully-selected markers visualized as color channels. Quantitative characterization of structures in acquired images often relies on automatic image analysis methods. Despite the success of deep learning methods in other vision applications, their potential for fluorescence image analysis remains underexploited. One reason lies in the considerable workload required to train accurate models, which are normally specific for a given combination of markers, and therefore applicable to a very restricted number of experimental settings. We herein propose Marker Sampling and Excite - a neural network approach with a modality sampling strategy and a novel attention module that together enable (i) flexible training with heterogeneous datasets with combinations of markers and (ii) successful utility of learned models on arbitrary subsets of markers prospectively. We show that our single neural network solution performs comparably to an upper bound scenario where an ensemble of many networks is naïvely trained for each possible marker combination separately. In addition, we demonstrate the feasibility of this framework in high-throughput biological analysis by revising a recent quantitative characterization of bone marrow vasculature in 3D confocal microscopy datasets and further confirm the validity of our approach on an additional, significantly different dataset of microvessels in fetal liver tissues. Not only can our work substantially ameliorate the use of deep learning in fluorescence microscopy analysis, but it can also be utilized in other fields with incomplete data acquisitions and missing modalities.

Assessing Cellular Hypoxic Status In Situ Within the Bone Marrow Microenvironment.

Hematopoietic stem cells are maintained and regulated in spatially confined microenvironments within the bone marrow, in which oxygen availability is hypothesized to be very limited. The hypoxic nature of HSC niches is proposed to play a fundamental role in the preservation of fundamental stem cell properties through the induction of a distinct glycolytic metabolic profile in HSCs. Thus, the capacity to determine oxygen levels or cellular oxygenation status in specific tissue locations is essential to deepen our understanding of HSC biology. We here describe a methodology to indirectly quantify the hypoxic status of individual cells in situ within histological sections of bone marrow tissues. We employ the well-characterized nitroimidazole probe, pimonidazole, which acts as an oxygen mimetic and irreversibly incorporates into cellular proteins only under hypoxic conditions. The use of fluorescently labeled antibodies that recognize pimonidazole epitopes then enables the indirect assessment of the intracellular hypoxic status and its relationship to cell positioning within the complex tissue topography of the bone marrow.

NG2 antigen is a therapeutic target for MLL-rearranged B-cell acute lymphoblastic leukemia.

B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with cure rates of ∼80%. MLL-rearranged (MLLr) B-ALL (MLLr-B-ALL) has, however, an unfavorable prognosis with common therapy refractoriness and early relapse, and therefore new therapeutic targets are needed for relapsed/refractory MLLr-B-ALL. MLLr leukemias are characterized by the specific expression of chondroitin sulfate proteoglycan-4, also known as neuron-glial antigen-2 (NG2). NG2 was recently shown involved in leukemia invasiveness and central nervous system infiltration in MLLr-B-ALL, and correlated with lower event-free survival (EFS). We here hypothesized that blocking NG2 may synergize with established induction therapy for B-ALL based on vincristine, glucocorticoids, and L-asparaginase (VxL). Using robust patient-derived xenograft (PDX) models, we found that NG2 is crucial for MLLr-B-ALL engraftment upon intravenous (i.v.) transplantation. In vivo blockade of NG2 using either chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significant mobilization of MLLr-B-ALL blasts from bone marrow (BM) to peripheral blood (PB) as demonstrated by cytometric and 3D confocal imaging analysis. When combined with either NG2 antagonist, VxL treatment achieved higher rates of complete remission, and consequently higher EFS and delayed time to relapse. Mechanistically, anti-NG2 MoAb induces neither antibody-dependent cell-mediated not complement-dependent cytotoxicity. NG2 blockade rather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to chemotherapy. We provide a proof of concept for NG2 as a therapeutic target for MLLr-B-ALL.

Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy.

Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.