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Adoption of high-content omic technologies in clinical studies, coupled with computational methods, has yielded an abundance of candidate biomarkers. However, translating such findings into bona fide clinical biomarkers remains challenging. To facilitate this process, we introduce Stabl, a general machine learning method that identifies a sparse, reliable set of biomarkers by integrating noise injection and a data-driven signal-to-noise threshold into multivariable predictive modeling. Evaluation of Stabl on synthetic datasets and five independent clinical studies demonstrates improved biomarker sparsity and reliability compared to commonly used sparsity-promoting regularization methods while maintaining predictive performance; it distills datasets containing 1,400-35,000 features down to 4-34 candidate biomarkers. Stabl extends to multi-omic integration tasks, enabling biological interpretation of complex predictive models, as it hones in on a shortlist of proteomic, metabolomic and cytometric events predicting labor onset, microbial biomarkers of pre-term birth and a pre-operative immune signature of post-surgical infections. Stabl is available at https://github.com/gregbellan/Stabl ., (© 2024. The Author(s).)

Oral squamous cell carcinoma (OSCC), a prevalent and aggressive neoplasm, poses a significant challenge due to poor prognosis and limited prognostic biomarkers. Leveraging highly multiplexed imaging mass cytometry, we investigated the tumor immune microenvironment (TIME) in OSCC biopsies, characterizing immune cell distribution and signaling activity at the tumor-invasive front. Our spatial subsetting approach standardized cellular populations by tissue zone, improving feature reproducibility and revealing TIME patterns accompanying loss-of-differentiation. Employing a machine-learning pipeline combining reliable feature selection with multivariable modeling, we achieved accurate histological grade classification (AUC = 0.88). Three model features correlated with clinical outcomes in an independent cohort: granulocyte MAPKAPK2 signaling at the tumor front, stromal CD4 + memory T cell size, and the distance of fibroblasts from the tumor border. This study establishes a robust modeling framework for distilling complex imaging data, uncovering sentinel characteristics of the OSCC TIME to facilitate prognostic biomarkers discovery for recurrence risk stratification and immunomodulatory therapy development., Competing Interests: C.M.S. is a scientific advisor to AstraZeneca plc, and is on the scientific advisory board of, has stock options in, and has received research funding from Enable Medicine, Inc. D.K.G., J.H., and B.G. are advisory board members at SurgeCare., (© 2023 The Author(s).)

Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation., (© 2023. The Author(s).)

Background: A critical size bone defect is a clinical scenario in which bone is lost or excised due to trauma, infection, tumor, or other causes, and cannot completely heal spontaneously. The most common treatment for this condition is autologous bone grafting to the defect site. However, autologous bone graft is often insufficient in quantity or quality for transplantation to these large defects. Recently, tissue engineering methods using mesenchymal stem cells (MSCs) have been proposed as an alternative treatment. However, the underlying biological principles and optimal techniques for tissue regeneration of bone using stem cell therapy have not been completely elucidated., Methods: In this study, we compare the early cellular dynamics of healing between bone graft transplantation and MSC therapy in a murine chronic femoral critical-size bone defect. We employ high-dimensional mass cytometry to provide a comprehensive view of the differences in cell composition, stem cell functionality, and immunomodulatory activity between these two treatment methods one week after transplantation., Results: We reveal distinct cell compositions among tissues from bone defect sites compared with original bone graft, show active recruitment of MSCs to the bone defect sites, and demonstrate the phenotypic diversity of macrophages and T cells in each group that may affect the clinical outcome., Conclusion: Our results provide critical data and future directions on the use of MSCs for treating critical size defects to regenerate bone. Translational Potential of this article: This study showed systematic comparisons of the cellular and immunomodulatory profiles among different interventions to improve the healing of the critical-size bone defect. The results provided potential strategies for designing robust therapeutic interventions for the unmet clinical need of treating critical-size bone defects., Competing Interests: The authors have no conflicts of interest relevant to this article., (© 2022 Published by Elsevier B.V. on behalf of Chinese Speaking Orthopaedic Society.)

The biological determinants underlying the range of coronavirus 2019 (COVID-19) clinical manifestations are not fully understood. Here, over 1,400 plasma proteins and 2,600 single-cell immune features comprising cell phenotype, endogenous signaling activity, and signaling responses to inflammatory ligands are cross-sectionally assessed in peripheral blood from 97 patients with mild, moderate, and severe COVID-19 and 40 uninfected patients. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identify and independently validate a multi-variate model classifying COVID-19 severity (multi-class area under the curve [AUC] training  = 0.799, p = 4.2e-6; multi-class AUC validation  = 0.773, p = 7.7e-6). Examination of informative model features reveals biological signatures of COVID-19 severity, including the dysregulation of JAK/STAT, MAPK/mTOR, and nuclear factor κB (NF-κB) immune signaling networks in addition to recapitulating known hallmarks of COVID-19. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for prevention and/or treatment of COVID-19 progression., Competing Interests: Declaration of interests S.D.B. has consulted for Regeneron, Sanofi, Novartis, and Janssen on topics unrelated to this study and owns stocks in AbCellera Biologics. The other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Approximately 1 in 4 pregnant women in the United States undergo labor induction. The onset and establishment of labor, particularly induced labor, is a complex and dynamic process influenced by multiple endocrine, inflammatory, and mechanical factors as well as obstetric and pharmacological interventions. The duration from labor induction to the onset of active labor remains unpredictable. Moreover, prolonged labor is associated with severe complications for the mother and her offspring, most importantly chorioamnionitis, uterine atony, and postpartum hemorrhage. While maternal immune system adaptations that are critical for the maintenance of a healthy pregnancy have been previously characterized, the role of the immune system during the establishment of labor is poorly understood. Understanding maternal immune adaptations during labor initiation can have important ramifications for predicting successful labor induction and labor complications in both induced and spontaneous types of labor. The aim of this study was to characterize labor-associated maternal immune system dynamics from labor induction to the start of active labor. Serial blood samples from fifteen participants were collected immediately prior to labor induction (baseline) and during the latent phase until the start of active labor. Using high-dimensional mass cytometry, a total of 1,059 single-cell immune features were extracted from each sample. A multivariate machine-learning method was employed to characterize the dynamic changes of the maternal immune system after labor induction until the establishment of active labor. A cross-validated linear sparse regression model (least absolute shrinkage and selection operator, LASSO) predicted the minutes since induction of labor with high accuracy (R = 0.86, p = 6.7e-15, RMSE = 277 min). Immune features most informative for the model included STAT5 signaling in central memory CD8 + T cells and pro-inflammatory STAT3 signaling responses across multiple adaptive and innate immune cell subsets. Our study reports a peripheral immune signature of labor induction, and provides important insights into biological mechanisms that may ultimately predict labor induction success as well as complications, thereby facilitating clinical decision-making to improve maternal and fetal well-being., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ando, Hédou, Feyaerts, Han, Ganio, Tsai, Peterson, Verdonk, Tsai, Marić, Wong, Angst, Aghaeepour, Stevenson, Blumenfeld, Sultan, Carvalho, Stelzer and Gaudillière.)

Although most causes of death and morbidity in premature infants are related to immune maladaptation, the premature immune system remains poorly understood. We provide a comprehensive single-cell depiction of the neonatal immune system at birth across the spectrum of viable gestational age (GA), ranging from 25 weeks to term. A mass cytometry immunoassay interrogated all major immune cell subsets, including signaling activity and responsiveness to stimulation. An elastic net model described the relationship between GA and immunome (R=0.85, p=8.75e-14), and unsupervised clustering highlighted previously unrecognized GA-dependent immune dynamics, including decreasing basal MAP-kinase/NFκB signaling in antigen presenting cells; increasing responsiveness of cytotoxic lymphocytes to interferon-α; and decreasing frequency of regulatory and invariant T cells, including NKT-like cells and CD8 + CD161 + T cells. Knowledge gained from the analysis of the neonatal immune landscape across GA provides a mechanistic framework to understand the unique susceptibility of preterm infants to both hyper-inflammatory diseases and infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Peterson, Hedou, Ganio, Stelzer, Feyaerts, Harbert, Adusumelli, Ando, Tsai, Tsai, Han, Ringle, Houghteling, Reiss, Lewis, Winn, Angst, Aghaeepour, Stevenson and Gaudilliere.)

Estimating the time of delivery is of high clinical importance because pre- and postterm deviations are associated with complications for the mother and her offspring. However, current estimations are inaccurate. As pregnancy progresses toward labor, major transitions occur in fetomaternal immune, metabolic, and endocrine systems that culminate in birth. The comprehensive characterization of maternal biology that precedes labor is key to understanding these physiological transitions and identifying predictive biomarkers of delivery. Here, a longitudinal study was conducted in 63 women who went into labor spontaneously. More than 7000 plasma analytes and peripheral immune cell responses were analyzed using untargeted mass spectrometry, aptamer-based proteomic technology, and single-cell mass cytometry in serial blood samples collected during the last 100 days of pregnancy. The high-dimensional dataset was integrated into a multiomic model that predicted the time to spontaneous labor [ R = 0.85, 95% confidence interval (CI) [0.79 to 0.89], P = 1.2 × 10 -40 , N = 53, training set; R = 0.81, 95% CI [0.61 to 0.91], P = 3.9 × 10 -7 , N = 10, independent test set]. Coordinated alterations in maternal metabolome, proteome, and immunome marked a molecular shift from pregnancy maintenance to prelabor biology 2 to 4 weeks before delivery. A surge in steroid hormone metabolites and interleukin-1 receptor type 4 that preceded labor coincided with a switch from immune activation to regulation of inflammatory responses. Our study lays the groundwork for developing blood-based methods for predicting the day of labor, anchored in mechanisms shared in preterm and term pregnancies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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Many diseases display unequal prevalence between sexes. The sex-specific immune response to both injury and persistent pain remains underexplored and would inform treatment paradigms. We utilized high-dimensional mass cytometry to perform a comprehensive analysis of phenotypic and functional immune system differences between male and female mice after orthopedic injury. Multivariate modeling of innate and adaptive immune cell responses after injury using an elastic net algorithm, a regularized regression method, revealed sex-specific divergence at 12 h and 7 days after injury with a stronger immune response to injury in females. At 12 h, females upregulated STAT3 signaling in neutrophils but downregulated STAT1 and STAT6 signals in T regulatory cells, suggesting a lack of engagement of immune suppression pathways by females. Furthermore, at 7 days females upregulated MAPK pathways (p38, ERK, NFkB) in CD4T memory cells, setting up a possible heightened immune memory of painful injury. Taken together, our findings provide the first comprehensive and functional analysis of sex-differences in the immune response to painful injury., (Copyright © 2020 Tawfik, Huck, Baca, Ganio, Haight, Culos, Ghaemi, Phongpreecha, Angst, Clark, Aghaeepour and Gaudilliere.)

Glucocorticoids (GC) are a controversial yet commonly used intervention in the clinical management of acute inflammatory conditions, including sepsis or traumatic injury. In the context of major trauma such as surgery, concerns have been raised regarding adverse effects from GC, thereby necessitating a better understanding of how GCs modulate the immune response. Here we report the results of a randomized controlled trial (NCT02542592) in which we employ a high-dimensional mass cytometry approach to characterize innate and adaptive cell signaling dynamics after a major surgery (primary outcome) in patients treated with placebo or methylprednisolone (MP). A robust, unsupervised bootstrap clustering of immune cell subsets coupled with random forest analysis shows profound (AUC = 0.92, p-value = 3.16E-8) MP-induced alterations of immune cell signaling trajectories, particularly in the adaptive compartments. By contrast, key innate signaling responses previously associated with pain and functional recovery after surgery, including STAT3 and CREB phosphorylation, are not affected by MP. These results imply cell-specific and pathway-specific effects of GCs, and also prompt future studies to examine GCs' effects on clinical outcomes likely dependent on functional adaptive immune responses.

Chronic periodontitis (ChP) is a prevalent inflammatory disease affecting 46% of the US population. ChP produces a profound local inflammatory response to dysbiotic oral microbiota that leads to destruction of alveolar bone and tooth loss. ChP is also associated with systemic illnesses, including cardiovascular diseases, malignancies, and adverse pregnancy outcomes. However, the mechanisms underlying these adverse health outcomes are poorly understood. In this prospective cohort study, we used a highly multiplex mass cytometry immunoassay to perform an in-depth analysis of the systemic consequences of ChP in patients before ( n = 28) and after ( n = 16) periodontal treatment. A high-dimensional analysis of intracellular signaling networks revealed immune system-wide dysfunctions differentiating patients with ChP from healthy controls. Notably, we observed exaggerated proinflammatory responses to Porphyromonas gingivalis -derived lipopolysaccharide in circulating neutrophils and monocytes from patients with ChP. Simultaneously, natural killer cell responses to inflammatory cytokines were attenuated. Importantly, the immune alterations associated with ChP were no longer detectable 3 wk after periodontal treatment. Our findings demarcate systemic and cell-specific immune dysfunctions in patients with ChP, which can be temporarily reversed by the local treatment of ChP. Future studies in larger cohorts are needed to test the boundaries of generalizability of our results.

Preeclampsia is one of the most severe pregnancy complications and a leading cause of maternal death. However, early diagnosis of preeclampsia remains a clinical challenge. Alterations in the normal immune adaptations necessary for the maintenance of a healthy pregnancy are central features of preeclampsia. However, prior analyses primarily focused on the static assessment of select immune cell subsets have provided limited information for the prediction of preeclampsia. Here, we used a high-dimensional mass cytometry immunoassay to characterize the dynamic changes of over 370 immune cell features (including cell distribution and functional responses) in maternal blood during healthy and preeclamptic pregnancies. We found a set of eight cell-specific immune features that accurately identified patients well before the clinical diagnosis of preeclampsia (median area under the curve (AUC) 0.91, interquartile range [0.82-0.92]). Several features recapitulated previously known immune dysfunctions in preeclampsia, such as elevated pro-inflammatory innate immune responses early in pregnancy and impaired regulatory T (Treg) cell signaling. The analysis revealed additional novel immune responses that were strongly associated with, and preceded the onset of preeclampsia, notably abnormal STAT5ab signaling dynamics in CD4 + T cell subsets (AUC 0.92, p = 8.0E-5). These results provide a global readout of the dynamics of the maternal immune system early in pregnancy and lay the groundwork for identifying clinically-relevant immune dysfunctions for the prediction and prevention of preeclampsia.

Stroke is a leading cause of cognitive impairment and dementia, but the mechanisms that underlie post-stroke cognitive decline are not well understood. Stroke produces profound local and systemic immune responses that engage all major innate and adaptive immune compartments. However, whether the systemic immune response to stroke contributes to long-term disability remains ill-defined. We used a single-cell mass cytometry approach to comprehensively and functionally characterize the systemic immune response to stroke in longitudinal blood samples from 24 patients over the course of 1 year and correlated the immune response with changes in cognitive functioning between 90 and 365 days post-stroke. Using elastic net regularized regression modelling, we identified key elements of a robust and prolonged systemic immune response to ischaemic stroke that occurs in three phases: an acute phase (Day 2) characterized by increased signal transducer and activator of transcription 3 (STAT3) signalling responses in innate immune cell types, an intermediate phase (Day 5) characterized by increased cAMP response element-binding protein (CREB) signalling responses in adaptive immune cell types, and a late phase (Day 90) by persistent elevation of neutrophils, and immunoglobulin M+ (IgM+) B cells. By Day 365 there was no detectable difference between these samples and those from an age- and gender-matched patient cohort without stroke. When regressed against the change in the Montreal Cognitive Assessment scores between Days 90 and 365 after stroke, the acute inflammatory phase Elastic Net model correlated with post-stroke cognitive trajectories (r = -0.692, Bonferroni-corrected P = 0.039). The results demonstrate the utility of a deep immune profiling approach with mass cytometry for the identification of clinically relevant immune correlates of long-term cognitive trajectories., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Early detection of maladaptive processes underlying pregnancy-related pathologies is desirable because it will enable targeted interventions ahead of clinical manifestations. The quantitative analysis of plasma proteins features prominently among molecular approaches used to detect deviations from normal pregnancy. However, derivation of proteomic signatures sufficiently predictive of pregnancy-related outcomes has been challenging. An important obstacle hindering such efforts were limitations in assay technology, which prevented the broad examination of the plasma proteome., Objective: The recent availability of a highly multiplexed platform affording the simultaneous measurement of 1310 plasma proteins opens the door for a more explorative approach. The major aim of this study was to examine whether analysis of plasma collected during gestation of term pregnancy would allow identifying a set of proteins that tightly track gestational age. Establishing precisely timed plasma proteomic changes during term pregnancy is a critical step in identifying deviations from regular patterns caused by fetal and maternal maladaptations. A second aim was to gain insight into functional attributes of identified proteins and link such attributes to relevant immunological changes., Study Design: Pregnant women participated in this longitudinal study. In 2 subsequent sets of 21 (training cohort) and 10 (validation cohort) women, specific blood specimens were collected during the first (7-14 weeks), second (15-20 weeks), and third (24-32 weeks) trimesters and 6 weeks postpartum for analysis with a highly multiplexed aptamer-based platform. An elastic net algorithm was applied to infer a proteomic model predicting gestational age. A bootstrapping procedure and piecewise regression analysis was used to extract the minimum number of proteins required for predicting gestational age without compromising predictive power. Gene ontology analysis was applied to infer enrichment of molecular functions among proteins included in the proteomic model. Changes in abundance of proteins with such functions were linked to immune features predictive of gestational age at the time of sampling in pregnancies delivering at term., Results: An independently validated model consisting of 74 proteins strongly predicted gestational age (P = 3.8 × 10 -14 , R = 0.97). The model could be reduced to 8 proteins without losing its predictive power (P = 1.7 × 10 -3 , R = 0.91). The 3 top ranked proteins were glypican 3, chorionic somatomammotropin hormone, and granulins. Proteins activating the Janus kinase and signal transducer and activator of transcription pathway were enriched in the proteomic model, chorionic somatomammotropin hormone being the top-ranked protein. Abundance of chorionic somatomammotropin hormone strongly correlated with signal transducer and activator of transcription-5 signaling activity in CD4 T cells, the endogenous cell-signaling event most predictive of gestational age., Conclusion: Results indicate that precisely timed changes in the plasma proteome during term pregnancy mirror a proteomic clock. Importantly, the combined use of several plasma proteins was required for accurate prediction. The exciting promise of such a clock is that deviations from its regular chronological profile may assist in the early diagnoses of pregnancy-related pathologies, and point to underlying pathophysiology. Functional analysis of the proteomic model generated the novel hypothesis that chrionic somatomammotropin hormone may critically regulate T-cell function during pregnancy., (Copyright © 2018. Published by Elsevier Inc.)

The maintenance of pregnancy relies on finely tuned immune adaptations. We demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using mass cytometry, the abundance and functional responses of all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. Cell signaling-based Elastic Net, a regularized regression method adapted from the elastic net algorithm, was developed to infer and prospectively validate a predictive model of interrelated immune events that accurately captures the chronology of pregnancy. Model components highlighted existing knowledge and revealed previously unreported biology, including a critical role for the interleukin-2-dependent STAT5ab signaling pathway in modulating T cell function during pregnancy. These findings unravel the precise timing of immunological events occurring during a term pregnancy and provide the analytical framework to identify immunological deviations implicated in pregnancy-related pathologies., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific, as they reduce infection rates in elective surgery, but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably, AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery, consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly, AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells, which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20-30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N 4 -acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4 + and CD8 + T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet + CD4 + T cells, CD8 + T cells, B cells, and CD56 lo CD16 + NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background: Recovery after surgery is highly variable. Risk-stratifying patients based on their predicted recovery profile will afford individualized perioperative management strategies. Recently, application of mass cytometry in patients undergoing hip arthroplasty revealed strong immune correlates of surgical recovery in blood samples collected shortly after surgery. However, the ability to interrogate a patient's immune state before surgery and predict recovery is highly desirable in perioperative medicine., Methods: To evaluate a patient's presurgical immune state, cell-type-specific intracellular signaling responses to ex vivo ligands (lipopolysaccharide, interleukin [IL]-6, IL-10, and IL-2/granulocyte macrophage colony-stimulating factor) were quantified by mass cytometry in presurgical blood samples. Selected ligands modulate signaling processes perturbed by surgery. Twenty-three cell surface and 11 intracellular markers were used for the phenotypic and functional characterization of major immune cell subsets. Evoked immune responses were regressed against patient-centered outcomes, contributing to protracted recovery including functional impairment, postoperative pain, and fatigue., Results: Evoked signaling responses varied significantly and defined patient-specific presurgical immune states. Eighteen signaling responses correlated significantly with surgical recovery parameters (|R| = 0.37 to 0.70; false discovery rate < 0.01). Signaling responses downstream of the toll-like receptor 4 in cluster of differentiation (CD) 14 monocytes were particularly strong correlates, accounting for 50% of observed variance. Immune correlates identified in presurgical blood samples mirrored correlates identified in postsurgical blood samples., Conclusions: Convergent findings in pre- and postsurgical analyses provide validation of reported immune correlates and suggest a critical role of the toll-like receptor 4 signaling pathway in monocytes for the clinical recovery process. The comprehensive assessment of patients' preoperative immune state is promising for predicting important recovery parameters and may lead to clinical tests using standard flow cytometry.

Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1)) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort., (© 2015 International Society for Advancement of Cytometry.)

Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery., (Copyright © 2014, American Association for the Advancement of Science.)