Combined protein and nucleic acid imaging reveals virus-dependent B cell and macrophage immunosuppression of tissue microenvironments.

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.

Human IL-10-producing B cells have diverse states that are induced from multiple B cell subsets.

Regulatory B cells (Bregs) suppress immune responses through the secretion of interleukin-10 (IL-10). This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. Here, we simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and time points using mass cytometry. Our analysis shows that multiple functional B cell subsets produce IL-10 and that no phenotype uniquely identifies IL-10+ B cells. Further, a significant portion of IL-10+ B cells co-express the pro-inflammatory cytokines IL-6 and tumor necrosis factor alpha (TNFα). Despite this heterogeneity, operationally tolerant liver transplant recipients have a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells compared with transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.

Multiplexed Ion Beam Imaging: Insights into Pathobiology.

Next-generation tools for multiplexed imaging have driven a new wave of innovation in understanding how single-cell function and tissue structure are interrelated. In previous work, we developed multiplexed ion beam imaging by time of flight, a highly multiplexed platform that uses secondary ion mass spectrometry to image dozens of antibodies tagged with metal reporters. As instrument throughput has increased, the breadth and depth of imaging data have increased as well. To extract meaningful information from these data, we have developed tools for cell identification, cell classification, and spatial analysis. In this review, we discuss these tools and provide examples of their application in various contexts, including ductal carcinoma in situ, tuberculosis, and Alzheimer's disease. We hope the synergy between multiplexed imaging and automated image analysis will drive a new era in anatomic pathology and personalized medicine wherein quantitative spatial signatures are used routinely for more accurate diagnosis, prognosis, and therapeutic selection.

MAUI (MBI Analysis User Interface)-An image processing pipeline for Multiplexed Mass Based Imaging.

Mass Based Imaging (MBI) technologies such as Multiplexed Ion Beam Imaging by time of flight (MIBI-TOF) and Imaging Mass Cytometry (IMC) allow for the simultaneous measurement of the expression levels of 40 or more proteins in biological tissue, providing insight into cellular phenotypes and organization in situ. Imaging artifacts, resulting from the sample, assay or instrumentation complicate downstream analyses and require correction by domain experts. Here, we present MBI Analysis User Interface (MAUI), a series of graphical user interfaces that facilitate this data pre-processing, including the removal of channel crosstalk, noise and antibody aggregates. Our software streamlines these steps and accelerates processing by enabling real-time and interactive parameter tuning across multiple images.

An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity.

B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27- early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.

Predictive Analytics and Modeling Employing Machine Learning Technology: The Next Step in Data Sharing, Analysis, and Individualized Counseling Explored With a Large, Prospective Prenatal Hydronephrosis Database.

OBJECTIVE: To explore the potential value of utilizing a commercially available cloud-based machine learning platform to predict surgical intervention in infants with prenatal hydronephrosis (HN). MATERIALS AND METHODS: A prospective prenatal HN database was uploaded into Microsoft Azure Machine Learning Studio. Probabilistic principal component analysis was employed for data imputation. Multiple clinical variables were included in two-class decision jungle and neural network for model training, using surgical intervention as the primary outcome. Models were scored and evaluated after a 70/30 split of the data. RESULTS: A total of 557 entries were included. The optimized model (decision jungle) achieved an area under the curve of 0.9, accuracy of 0.87, and precision of 0.80, employing a threshold of 0.5 to predict surgery. Average time to train, score and evaluate the model was 5 seconds. The predictive model was deployed as a web service in 35 seconds, generating a unique API key for app and webpage development. Individualized prediction based on the included variables was deployed as a web-based and batch execution Excel file in less than one minute. CONCLUSION: This cloud-based ML technology allows easy building, deployment, and sharing of predictive analytics solutions. Using prenatal HN as an example, we propose an opportunity to address contemporary challenges with data analysis, reporting a creative solution that moves beyond the current standard.

Human Bronchial Epithelial Cell-derived Factors from Severe Asthmatic Subjects Stimulate Eosinophil Differentiation.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

Comparative Outcome Analysis of Children Who Underwent Pyeloplasty for Ureteropelvic Junction Obstruction Associated With or Without Supranormal Differential Renal Function.

OBJECTIVE: To compare pyeloplasty outcomes in children with and without "supra-normal" differential renal function (SNDRF) defined as >55% differential renal function (DRF) in children with ureteropelvic junction obstruction. METHODS: Our prospectively collected pyeloplasty database (2008-2015) was reviewed (n = 151). A total of 140/151 (93%) patients had preoperative renograms and 26/140 (19%) were found to have SNDRF (DRF ≥ 55%). Of 151 patients, 51 (34%) had pre- and postoperative renograms allowing determination of change ≥5% in function. After excluding 2 patients with solitary kidneys, a total of 49 patients defined the study group. RESULTS: Of 49 patients, 12 had SNDRF and 37 did not. Baseline characteristics were similar including mean age at surgery (47.3 months vs 45.4 months) and time to surgery (8.7 months vs 9.8 months). Mean preoperative anteroposterior diameter was significantly different between groups (23.2 mm vs 31.0 mm; P = .04), but postoperative was similar (9.0 mm vs 12.1 mm; P = .14). Mean preoperative DRF was 60.2% in the SNDRF group vs 44.3% in the non-SNDRF. Mean postoperative DRF was 52.4% and 45.3%, respectively (P = .04). There were 9/12 (75%) SNDRF patients who experienced ≥5% loss in function compared to 2/37 (5%) in the non-SNDRF group (P < .01). CONCLUSION: Three-quarters of SNDRF patients demonstrated a decline of ≥5%DRF postoperatively when compared to non-SNDRF. This finding may not reflect true elevated renal function, but rather hyperfiltration in the setting of obstruction, which-if unrecognized as such-could result in postponing an otherwise beneficial surgical intervention.

Allergen-induced Changes in Bone Marrow and Airway Dendritic Cells in Subjects with Asthma.

RATIONALE: Dendritic cells (DCs) are antigen-presenting cells essential for the initiation of T-cell responses. Allergen inhalation increases the number of airway DCs and the release of epithelial-derived cytokines, such as IL-33 and thymic stromal lymphopoietin (TSLP), that activate DCs. OBJECTIVES: To examine the effects of inhaled allergen on bone marrow production of DCs and their trafficking into the airways in subjects with allergic asthma, and to examine IL-33 and TSPL receptor expression on DCs. METHODS: Bone marrow, peripheral blood, bronchoalveolar lavage (BAL), and bronchial biopsies were obtained before and after inhalation of diluent and allergen from subjects with asthma that develop allergen-induced dual responses. Classical DCs (cDCs) were cultured from bone marrow CD34(+) cells. cDC1s, cDC2s, and plasmacytoid DCs were measured in bone marrow aspirates, peripheral blood, and BAL by flow cytometry, and cDCs were quantified in bronchial biopsies by immunofluorescence staining. MEASUREMENTS AND MAIN RESULTS: Inhaled allergen increased the number of cDCs grown from bone marrow progenitors, and cDCs and plasmacytoid DCs in bone marrow aspirates 24 hours after allergen. Allergen also increased the expression of the TSLP receptor, but not the IL-33 receptor, on bone marrow DCs. Finally, inhaled allergen increased the percentage of cDC1s and cDC2s in BAL but only cDC2s in bronchial tissues. CONCLUSIONS: Inhaled allergen increases DCs in bone marrow and trafficking of DCs into the airway, which is associated with the development airway inflammation in subjects with allergic asthma. Inhaled allergen challenge also increases expression of TSLP, but not IL-33, receptors on bone marrow DCs.

Thymic stromal lymphopoietin activation of basophils in patients with allergic asthma is IL-3 dependent.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) released after antigenic stimulation of allergic asthmatic airways is a key initiator of type 2 inflammation. Basophils are important effectors of allergic inflammation in the airways. Murine basophils have been shown to respond to TSLP independently of IL-3 by increasing functional thymic stromal lymphopoietin receptor (TSLPR) expression. OBJECTIVE: The purpose of this study was to investigate the effect of TSLP stimulation on human basophil function. METHODS: Ten patients with mild allergic asthma underwent diluent and allergen inhalation challenges. Peripheral blood and sputum samples were collected at baseline and 7 and 24 hours after challenge, and bone marrow samples were collected at baseline and 24 hours after challenge to measure basophil TSLPR expression. In vitro experiments were conducted on purified human basophils to measure the effect of TSLP on degranulation, expression of activation markers and TH2 cytokines, and eotaxin-induced shape change. RESULTS: Allergen inhalation increased basophil numbers in the airways and significantly upregulated the expression of activation markers, TH2 intracellular cytokines, and receptors for TSLP, IL-3, and eotaxin in blood, bone marrow, and sputum basophils. In vitro stimulation with TSLP primed basophil migration to eotaxin and induced rapid and sustained basophil activation mediated directly through TSLPR and indirectly through an IL-3-mediated basophil autocrine loop. Basophils responded to TSLP at a similar magnitude and potency as the well-described basophil-activating stimuli IL-3 and anti-IgE. CONCLUSION: Our findings indicate that basophil activation during early- and late-phase responses to inhaled allergen might be driven at least in part by TSLP.

Thymic stromal lymphopoietin and IL-33 modulate migration of hematopoietic progenitor cells in patients with allergic asthma.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) and IL-33 are considered important initiators of type 2 immunity. In asthmatic patients allergic inflammatory responses are associated with increased lung homing of bone marrow-derived CD34(+) hematopoietic progenitor cells (HPCs), which include eosinophil lineage-committed progenitor cells. In this study we investigated the role of TSLP and IL-33 in the recruitment of progenitor cells to the airways in asthmatic subjects. OBJECTIVES: We sought (i) to examine the effect of allergen inhalation challenge on expression levels of receptors for TSLP (thymic stromal lymphopoietin receptor [TSLPR] and CD127) and IL-33 (ST2) and (ii) investigate the functional effects of these cytokines on HPCs. METHODS: Consenting patients with mild atopic asthma (n = 19) with an FEV1 of 70% or greater and methacholine PC20 of 16 mg/mL or less were recruited. Blood- and sputum-extracted progenitors were phenotyped by flow cytometry before and 24 hours after allergen challenge. Functional responses, including cytokine production and migration to TSLP and IL-33, were assessed in vitro. RESULTS: Significant increases in mature eosinophil, HPC, and eosinophil lineage-committed progenitor cell counts in sputum were observed 24 hours after allergen and were associated with a significant allergen-induced increase in HPCs expressing TSLPR, CD127, and ST2. Pre-exposure to TSLP and IL-33 primed the migration of HPCs to a potent progenitor cell chemoattractant, stromal cell-derived factor 1α (CXCL12). Incubation with TSLP and IL-33 stimulated significant production of IL-5 and IL-13, but not IL-4, by HPCs. This priming effect was inhibited by blocking antibodies to TSLPR and ST2, respectively, and IL-13 receptor α1 in both scenarios. CONCLUSIONS: In allergic asthmatic responses increased lung homing of HPCs may be orchestrated by TSLP and IL-33 through an IL-13-dependent axis.