spillR: spillover compensation in mass cytometry data.

MOTIVATION: Channel interference in mass cytometry can cause spillover and may result in miscounting of protein markers. Chevrier et al. introduce an experimental and computational procedure to estimate and compensate for spillover implemented in their R package CATALYST. They assume spillover can be described by a spillover matrix that encodes the ratio between the signal in the unstained spillover receiving and stained spillover emitting channel. They estimate the spillover matrix from experiments with beads. We propose to skip the matrix estimation step and work directly with the full bead distributions. We develop a nonparametric finite mixture model and use the mixture components to estimate the probability of spillover. Spillover correction is often a pre-processing step followed by downstream analyses, and choosing a flexible model reduces the chance of introducing biases that can propagate downstream. RESULTS: We implement our method in an R package spillR using expectation-maximization to fit the mixture model. We test our method on simulated, semi-simulated, and real data from CATALYST. We find that our method compensates low counts accurately, does not introduce negative counts, avoids overcompensating high counts, and preserves correlations between markers that may be biologically meaningful. AVAILABILITY AND IMPLEMENTATION: Our new R package spillR is on bioconductor at bioconductor.org/packages/spillR. All experiments and plots can be reproduced by compiling the R markdown file spillR_paper.Rmd at github.com/ChristofSeiler/spillR_paper.

CytoGLMM: conditional differential analysis for flow and mass cytometry experiments.

BACKGROUND: Flow and mass cytometry are important modern immunology tools for measuring expression levels of multiple proteins on single cells. The goal is to better understand the mechanisms of responses on a single cell basis by studying differential expression of proteins. Most current data analysis tools compare expressions across many computationally discovered cell types. Our goal is to focus on just one cell type. Our narrower field of application allows us to define a more specific statistical model with easier to control statistical guarantees. RESULTS: Differential analysis of marker expressions can be difficult due to marker correlations and inter-subject heterogeneity, particularly for studies of human immunology. We address these challenges with two multiple regression strategies: a bootstrapped generalized linear model and a generalized linear mixed model. On simulated datasets, we compare the robustness towards marker correlations and heterogeneity of both strategies. For paired experiments, we find that both strategies maintain the target false discovery rate under medium correlations and that mixed models are statistically more powerful under the correct model specification. For unpaired experiments, our results indicate that much larger patient sample sizes are required to detect differences. We illustrate the CytoGLMM R package and workflow for both strategies on a pregnancy dataset. CONCLUSION: Our approach to finding differential proteins in flow and mass cytometry data reduces biases arising from marker correlations and safeguards against false discoveries induced by patient heterogeneity.

Differential Induction of IFN-α and Modulation of CD112 and CD54 Expression Govern the Magnitude of NK Cell IFN-γ Response to Influenza A Viruses.

In human and murine studies, IFN-γ is a critical mediator immunity to influenza. IFN-γ production is critical for viral clearance and the development of adaptive immune responses, yet excessive production of IFN-γ and other cytokines as part of a cytokine storm is associated with poor outcomes of influenza infection in humans. As NK cells are the main population of lung innate immune cells capable of producing IFN-γ early in infection, we set out to identify the drivers of the human NK cell IFN-γ response to influenza A viruses. We found that influenza triggers NK cells to secrete IFN-γ in the absence of T cells and in a manner dependent upon signaling from both cytokines and receptor-ligand interactions. Further, we discovered that the pandemic A/California/07/2009 (H1N1) strain elicits a seven-fold greater IFN-γ response than other strains tested, including a seasonal A/Victoria/361/2011 (H3N2) strain. These differential responses were independent of memory NK cells. Instead, we discovered that the A/Victoria/361/2011 influenza strain suppresses the NK cell IFN-γ response by downregulating NK-activating ligands CD112 and CD54 and by repressing the type I IFN response in a viral replication-dependent manner. In contrast, the A/California/07/2009 strain fails to repress the type I IFN response or to downregulate CD54 and CD112 to the same extent, which leads to the enhanced NK cell IFN-γ response. Our results indicate that influenza implements a strain-specific mechanism governing NK cell production of IFN-γ and identifies a previously unrecognized influenza innate immune evasion strategy.

NKp30 and NKG2D contribute to natural killer recognition of HIV-infected cells.

Natural killer (NK) cells respond rapidly in early HIV-1 infection. HIV-1 prevention and control strategies harnessing NK cells could be enabled by mechanistic understanding of how NK cells recognize HIV-infected T cells. Here, we profiled the phenotype of human primary NK cells responsive to autologous HIV-1-infected CD4 + T cells in vitro. We characterized the patterns of NK cell ligand expression on CD4 + T cells at baseline and after infection with a panel of transmitted/founder HIV-1 strains to identify key receptor-ligand pairings. CRISPR editing of CD4 + T cells to knockout the NKp30 ligand B7-H6, or the NKG2D ligands MICB or ULBP2 reduced NK cell responses to HIV-infected cells in some donors. In contrast, overexpression of NKp30 or NKG2D in NK cells enhanced their targeting of HIV-infected cells. Collectively, we identified receptor-ligand pairs including NKp30:B7-H6 and NKG2D:MICB/ULBP2 that contribute to NK cell recognition of HIV-infected cells.

Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs.

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

Validity of an automatic measure protocol in distal femur for allograft selection from a three-dimensional virtual bone bank system.

Osteoarticular allograft is one possible treatment in wide surgical resections with large defects. Performing best osteoarticular allograft selection is of great relevance for optimal exploitation of the bone databank, good surgery outcome and patient's recovery. Current approaches are, however, very time consuming hindering these points in practice. We present a validation study of a software able to perform automatic bone measurements used to automatically assess the distal femur sizes across a databank. 170 distal femur surfaces were reconstructed from CT data and measured manually using a size measure protocol taking into account the transepicondyler distance (A), anterior-posterior distance in medial condyle (B) and anterior-posterior distance in lateral condyle (C). Intra- and inter-observer studies were conducted and regarded as ground truth measurements. Manual and automatic measures were compared. For the automatic measurements, the correlation coefficients between observer one and automatic method, were of 0.99 for A measure and 0.96 for B and C measures. The average time needed to perform the measurements was of 16 h for both manual measurements, and of 3 min for the automatic method. Results demonstrate the high reliability and, most importantly, high repeatability of the proposed approach, and considerable speed-up on the planning.

Natural killer cell phenotype is altered in HIV-exposed seronegative women.

Highly exposed seronegative (HESN) individuals present a unique setting to study mechanisms of protection against HIV acquisition. As natural killer (NK) cell activation and function have been implicated as a correlate of protection in HESN individuals, we sought to better understand the features of NK cells that may confer protection. We used mass cytometry to phenotypically profile NK cells from a cohort of Beninese sex workers and healthy controls. We found that NK cells from HESN women had increased expression of NKG2A, NKp30 and LILRB1, as well as the Fc receptor CD16, and decreased expression of DNAM-1, CD94, Siglec-7, and NKp44. Using functional assessments of NK cells from healthy donors against autologous HIV-infected CD4+ T cells, we observed that NKp30+ and Siglec-7+ cells had improved functional activity. Further, we found that NK cells from HESN women trended towards increased antibody-dependent cellular cytotoxicity (ADCC) activity; this activity correlated with increased CD16 expression. Overall, we identify features of NK cells in HESN women that may contribute to protection from HIV infection. Follow up studies with larger cohorts are warranted to confirm these findings.

Influenza-Induced Interferon Lambda Response Is Associated With Longer Time to Delivery Among Pregnant Kenyan Women.

Specific causes of preterm birth remain unclear. Several recent studies have suggested that immune changes during pregnancy are associated with the timing of delivery, yet few studies have been performed in low-income country settings where the rates of preterm birth are the highest. We conducted a retrospective nested case-control evaluation within a longitudinal study among HIV-uninfected pregnant Kenyan women. To characterize immune function in these women, we evaluated unstimulated and stimulated peripheral blood mononuclear cells in vitro with the A/California/2009 strain of influenza to understand the influenza-induced immune response. We then evaluated transcript expression profiles using the Affymetrix Human GeneChip Transcriptome Array 2.0. Transcriptional profiles of sufficient quality for analysis were obtained from 54 women; 19 of these women delivered <34 weeks and were defined as preterm cases and 35 controls delivered >37 weeks. The median time to birth from sample collection was 13 weeks. No transcripts were significantly associated with preterm birth in a case-control study of matched term and preterm birth (n = 42 women). In the influenza-stimulated samples, expression of IFNL1 was associated with longer time to delivery-the amount of time between sample collection and delivery (n = 54 women). A qPCR analysis confirmed that influenza-induced IFNL expression was associated with longer time to delivery. These data indicate that during pregnancy, ex vivo influenza stimulation results in altered transcriptional response and is associated with time to delivery in cohort of women residing in an area with high preterm birth prevalence.

Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry.

Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug's therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.

TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells.

OBJECTIVE: Our objective was to investigate the mechanisms that govern natural killer (NK)-cell responses to HIV, with a focus on specific receptor--ligand interactions involved in HIV recognition by NK cells. DESIGN AND METHODS: We first performed a mass cytometry-based screen of NK-cell receptor expression patterns in healthy controls and HIV individuals. We then focused mechanistic studies on the expression and function of T cell immunoreceptor with Ig and ITIM domains (TIGIT). RESULTS: The mass cytometry screen revealed that TIGIT is upregulated on NK cells of untreated HIV women, but not in antiretroviral-treated women. TIGIT is an inhibitory receptor that is thought to mark exhausted NK cells; however, blocking TIGIT did not improve anti-HIV NK-cell responses. In fact, the TIGIT ligands CD112 and CD155 were not upregulated on CD4 T cells in vitro or in vivo, providing an explanation for the lack of benefit from TIGIT blockade. TIGIT expression marked a unique subset of NK cells that express significantly higher levels of NK-cell-activating receptors (DNAM-1, NTB-A, 2B4, CD2) and exhibit a mature/adaptive phenotype (CD57, NKG2C, LILRB1, FcRγ, Syk). Furthermore, TIGIT NK cells had increased responses to mock-infected and HIV-infected autologous CD4 T cells, and to PMA/ionomycin, cytokine stimulation and the K562 cancer cell line. CONCLUSION: TIGIT expression is increased on NK cells from untreated HIV individuals. Although TIGIT does not participate directly to the response to HIV-infected cells, it marks a population of mature/adaptive NK cells with increased functional responses.

Pregnancy-Induced Alterations in NK Cell Phenotype and Function.

Pregnant women are particularly susceptible to complications of influenza A virus infection, which may result from pregnancy-induced changes in the function of immune cells, including natural killer (NK) cells. To better understand NK cell function during pregnancy, we assessed the ability of the two main subsets of NK cells, CD56dim, and CD56bright NK cells, to respond to influenza-virus infected cells and tumor cells. During pregnancy, CD56dim and CD56bright NK cells displayed enhanced functional responses to both infected and tumor cells, with increased expression of degranulation markers and elevated frequency of NK cells producing IFN-γ. To better understand the mechanisms driving this enhanced function, we profiled CD56dim and CD56bright NK cells from pregnant and non-pregnant women using mass cytometry. NK cells from pregnant women displayed significantly increased expression of several functional and activation markers such as CD38 on both subsets and NKp46 on CD56dim NK cells. NK cells also displayed diminished expression of the chemokine receptor CXCR3 during pregnancy. Overall, these data demonstrate that functional and phenotypic shifts occur in NK cells during pregnancy that can influence the magnitude of the immune response to both infections and tumors.

Multi-Table Differential Correlation Analysis of Neuroanatomical and Cognitive Interactions in Turner Syndrome.

Girls and women with Turner syndrome (TS) have a completely or partially missing X chromosome. Extensive studies on the impact of TS on neuroanatomy and cognition have been conducted. The integration of neuroanatomical and cognitive information into one consistent analysis through multi-table methods is difficult and most standard tests are underpowered. We propose a new two-sample testing procedure that compares associations between two tables in two groups. The procedure combines multi-table methods with permutation tests. In particular, we construct cluster size test statistics that incorporate spatial dependencies. We apply our new procedure to a newly collected dataset comprising of structural brain scans and cognitive test scores from girls with TS and healthy control participants (age and sex matched). We measure neuroanatomy with Tensor-Based Morphometry (TBM) and cognitive function with Wechsler IQ and NEuroPSYchological tests (NEPSY-II). We compare our multi-table testing procedure to a single-table analysis. Our new procedure reports differential correlations between two voxel clusters and a wide range of cognitive tests whereas the single-table analysis reports no differences. Our findings are consistent with the hypothesis that girls with TS have a different brain-cognition association structure than healthy controls.

Multivariate Heteroscedasticity Models for Functional Brain Connectivity.

Functional brain connectivity is the co-occurrence of brain activity in different areas during resting and while doing tasks. The data of interest are multivariate timeseries measured simultaneously across brain parcels using resting-state fMRI (rfMRI). We analyze functional connectivity using two heteroscedasticity models. Our first model is low-dimensional and scales linearly in the number of brain parcels. Our second model scales quadratically. We apply both models to data from the Human Connectome Project (HCP) comparing connectivity between short and conventional sleepers. We find stronger functional connectivity in short than conventional sleepers in brain areas consistent with previous findings. This might be due to subjects falling asleep in the scanner. Consequently, we recommend the inclusion of average sleep duration as a covariate to remove unwanted variation in rfMRI studies. A power analysis using the HCP data shows that a sample size of 40 detects 50% of the connectivity at a false discovery rate of 20%. We provide implementations using R and the probabilistic programming language Stan.

Image-based vs. mesh-based statistical appearance models of the human femur: implications for finite element simulations.

Statistical appearance models have recently been introduced in bone mechanics to investigate bone geometry and mechanical properties in population studies. The establishment of accurate anatomical correspondences is a critical aspect for the construction of reliable models. Depending on the representation of a bone as an image or a mesh, correspondences are detected using image registration or mesh morphing. The objective of this study was to compare image-based and mesh-based statistical appearance models of the femur for finite element (FE) simulations. To this aim, (i) we compared correspondence detection methods on bone surface and in bone volume; (ii) we created an image-based and a mesh-based statistical appearance models from 130 images, which we validated using compactness, representation and generalization, and we analyzed the FE results on 50 recreated bones vs. original bones; (iii) we created 1000 new instances, and we compared the quality of the FE meshes. Results showed that the image-based approach was more accurate in volume correspondence detection and quality of FE meshes, whereas the mesh-based approach was more accurate for surface correspondence detection and model compactness. Based on our results, we recommend the use of image-based statistical appearance models for FE simulations of the femur.

Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation.

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of morphology-based approaches.

Spatio-temporal dimension reduction of cardiac motion for group-wise analysis and statistical testing.

Given the observed abnormal motion dynamics of patients with heart conditions, quantifying cardiac motion in both normal and pathological cases can provide useful insights for therapy planning. In order to be able to analyse the motion over multiple subjects in a robust manner, it is desirable to represent the motion by a low number of parameters. We propose a reduced order cardiac motion model, reduced in space through a polyaffine model, and reduced in time by statistical model order reduction. The method is applied to a data-set of synthetic cases with known ground truth to validate the accuracy of the left ventricular motion tracking, and to validate a patient-specific reduced-order motion model. Population-based statistics are computed on a set of 15 healthy volunteers to obtain separate spatial and temporal bases. Results demonstrate that the reduced model can efficiently detect abnormal motion patterns and even allowed to retrospectively reveal abnormal unnoticed motion within the control subjects.

Simultaneous multiscale polyaffine registration by incorporating deformation statistics.

Locally affine (polyaffine) image registration methods capture intersubject non-linear deformations with a low number of parameters, while providing an intuitive interpretation for clinicians. Considering the mandible bone, anatomical shape differences can be found at different scales, e.g. left or right side, teeth, etc. Classically, sequential coarse to fine registration are used to handle multiscale deformations, instead we propose a simultaneous optimization of all scales. To avoid local minima we incorporate a prior on the polyaffine transformations. This kind of groupwise registration approach is natural in a polyaffine context, if we assume one configuration of regions that describes an entire group of images, with varying transformations for each region. In this paper, we reformulate polyaffine deformations in a generative statistical model, which enables us to incorporate deformation statistics as a prior in a Bayesian setting. We find optimal transformations by optimizing the maximum a posteriori probability. We assume that the polyaffine transformations follow a normal distribution with mean and concentration matrix. Parameters of the prior are estimated from an initial coarse to fine registration. Knowing the region structure, we develop a blockwise pseudoinverse to obtain the concentration matrix. To our knowledge, we are the first to introduce simultaneous multiscale optimization through groupwise polyaffine registration. We show results on 42 mandible CT images.

Capturing the multiscale anatomical shape variability with polyaffine transformation trees.

Mandible fractures are classified depending on their location. In clinical practice, locations are grouped into regions at different scales according to anatomical, functional and esthetic considerations. Implant design aims at defining the optimal implant for each patient. Emerging population-based techniques analyze the anatomical variability across a population and perform statistical analysis to identify an optimal set of implants. Current efforts are focused on finding clusters of patients with similar characteristics and designing one implant for each cluster. Ideally, the description of anatomical variability is directly connected to the clinical regions. This connection is what we present here, by introducing a new registration method that builds upon a tree of locally affine transformations that describes variability at different scales. We assess the accuracy of our method on 146 CT images of femurs. Two medical experts provide the ground truth by manually measuring six landmarks. We illustrate the clinical importance of our method by clustering 43 CT images of mandibles for implant design. The presented method does not require any application-specific input, which makes it attractive for the analysis of other multiscale anatomical structures. At the core of our new method lays the introduction of a new basis for stationary velocity fields. This basis has very close links to anatomical substructures. In the future, this method has the potential to discover the hidden and possibly sparse structure of the anatomy.

Population-based design of mandibular plates based on bone quality and morphology.

In this paper we present a new population-based implant design methodology, which advances the state-of-the-art approaches by combining shape and bone quality information into the design strategy. The method enhances the mechanical stability of the fixation and reduces the intra-operative in-plane bending which might impede the functionality of the locking mechanism. The method is presented for the case of mandibular locking fixation plates, where the mandibular angle and the bone quality at screw locations are taken into account. Using computational anatomy techniques, the method automatically derives, from a set of computed tomography images, the mandibular angle and the bone thickness and intensity values at the path of every screw. An optimisation strategy is then used to optimise the two parameters of plate angle and screw position. Results for the new design are presented along with a comparison with a commercially available mandibular locking fixation plate. A statistically highly significant improvement was observed. Our experiments allowed us to conclude that an angle of 126 degrees and a screw separation of 8 mm is a more suitable design than the standard 120 degrees and 9 mm.

Statistical model based shape prediction from a combination of direct observations and various surrogates: application to orthopaedic research.

In computer-assisted orthopaedic surgery, recovering three-dimensional patient-specific anatomy from incomplete information has been focus of interest due to several factors such as less invasive surgical procedures, reduced radiation doses, and rapid intra-operative updates of the anatomy. The aim of this paper is to report results obtained combining statistical shape modeling and multivariate regression techniques for predicting bone shape from clinically and surgically relevant predictors, including sparse observations of the bone surface but also morphometric and anthropometric information. Different state of the art methods such as partial least square regression, principal component regression, canonical correlation analysis, and non-parametric kernel-based regression are compared. Clinically relevant surrogate variables and combinations are investigated on a database of 142 femur and 154 tibia shapes obtained from CT images. The results are evaluated using cross validation to quantify the prediction error. The proposed approach enables to characterize the added value of different predictors in a quantitative and localized fashion. Results indicate that complementary sources of information can be efficiently exploited to improve the accuracy of shape prediction.