Publications by authors named "Gabriela K Fragiadakis"

24 Publications

  • Page 1 of 1

A "data sharing trust" model for rapid, collaborative science.

Cell 2021 Feb;184(3):566-570

ImmunoX Initiative, Department of Microbiology and Immunology, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0511, USA; UCSF CoLabs, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0511, USA; Division of Rheumatology, Department of Medicine, University of California, San Francisco, 513 Parnassus Ave, San Francisco, CA 94143-0511, USA. Electronic address:

Complex datasets provide opportunities for discoveries beyond their initial scope. Effective and rapid data sharing and management practices are crucial to realize this potential; however, they are harder to implement than post-publication access. Here, we introduce the concept of a "data sharing trust" to maximize the value of large datasets.
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http://dx.doi.org/10.1016/j.cell.2021.01.006DOI Listing
February 2021

Global absence and targeting of protective immune states in severe COVID-19.

Nature 2021 Mar 25;591(7848):124-130. Epub 2021 Jan 25.

Department of Pathology, University of California San Francisco, San Francisco, CA, USA.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs) across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.
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http://dx.doi.org/10.1038/s41586-021-03234-7DOI Listing
March 2021

dittoSeq: Universal User-Friendly Single-Cell and Bulk RNA Sequencing Visualization Toolkit.

Bioinformatics 2020 Dec 12. Epub 2020 Dec 12.

Bakar Computational Health Sciences Institute University of California, San Francisco, San Francisco, CA, USA.

Summary: A visualization suite for major forms of bulk and single-cell RNAseq data in R. dittoSeq is color blindness-friendly by default, robustly documented to power ease-of-use, and allows highly customizable generation of both daily-use and publication-quality figures.

Availability And Implementation: dittoSeq is an R package available through Bioconductor via an open source MIT license.

Supplementary Information: Supplementary Code and figures are available at Bioinformatics online. Full vignettes are available through Bioconductor, https://bioconductor.org/packages/dittoSeq/, and github, github.com/dtm2451/dittoSeq/.
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http://dx.doi.org/10.1093/bioinformatics/btaa1011DOI Listing
December 2020

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

bioRxiv 2020 Oct 29. Epub 2020 Oct 29.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.

One Sentence Summary: In severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.
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http://dx.doi.org/10.1101/2020.10.28.359935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605559PMC
October 2020

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

Res Sq 2020 Oct 28. Epub 2020 Oct 28.

Department of Pathology, San Francisco, 513 Parnassus Ave, HSW512, San Francisco, CA 94143-0511, USA.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a wholeblood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferonstimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and autodirected antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense.
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http://dx.doi.org/10.21203/rs.3.rs-97042/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605560PMC
October 2020

Landscape of coordinated immune responses to H1N1 challenge in humans.

J Clin Invest 2020 11;130(11):5800-5816

Department of Pathology and.

Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.
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http://dx.doi.org/10.1172/JCI137265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598057PMC
November 2020

Long-term dietary intervention reveals resilience of the gut microbiota despite changes in diet and weight.

Am J Clin Nutr 2020 06;111(6):1127-1136

Stanford Prevention Research Center, Department of Medicine, Stanford School of Medicine, Stanford, CA, USA.

Background: With the rising rates of obesity and associated metabolic disorders, there is a growing need for effective long-term weight-loss strategies, coupled with an understanding of how they interface with human physiology. Interest is growing in the potential role of gut microbes as they pertain to responses to different weight-loss diets; however, the ways that diet, the gut microbiota, and long-term weight loss influence one another is not well understood.

Objectives: Our primary objective was to determine if baseline microbiota composition or diversity was associated with weight-loss success. A secondary objective was to track the longitudinal associations of changes to lower-carbohydrate or lower-fat diets and concomitant weight loss with the composition and diversity of the gut microbiota.

Methods: We used 16S ribosomal RNA gene amplicon sequencing to profile microbiota composition over a 12-mo period in 49 participants as part of a larger randomized dietary intervention study of participants consuming either a healthy low-carbohydrate or a healthy low-fat diet.

Results: While baseline microbiota composition was not predictive of weight loss, each diet resulted in substantial changes in the microbiota 3-mo after the start of the intervention; some of these changes were diet specific (14 taxonomic changes specific to the healthy low-carbohydrate diet, 12 taxonomic changes specific to the healthy low-fat diet) and others tracked with weight loss (7 taxonomic changes in both diets). After these initial shifts, the microbiota returned near its original baseline state for the remainder of the intervention, despite participants maintaining their diet and weight loss for the entire study.

Conclusions: These results suggest a resilience to perturbation of the microbiota's starting profile. When considering the established contribution of obesity-associated microbiotas to weight gain in animal models, microbiota resilience may need to be overcome for long-term alterations to human physiology. This trial was registered at clinicaltrials.gov as NCT01826591.
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http://dx.doi.org/10.1093/ajcn/nqaa046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266695PMC
June 2020

FLOW-MAP: a graph-based, force-directed layout algorithm for trajectory mapping in single-cell time course datasets.

Nat Protoc 2020 02 13;15(2):398-420. Epub 2020 Jan 13.

Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA.

High-dimensional single-cell technologies present new opportunities for biological discovery, but the complex nature of the resulting datasets makes it challenging to perform comprehensive analysis. One particular challenge is the analysis of single-cell time course datasets: how to identify unique cell populations and track how they change across time points. To facilitate this analysis, we developed FLOW-MAP, a graphical user interface (GUI)-based software tool that uses graph layout analysis with sequential time ordering to visualize cellular trajectories in high-dimensional single-cell datasets obtained from flow cytometry, mass cytometry or single-cell RNA sequencing (scRNAseq) experiments. Here we provide a detailed description of the FLOW-MAP algorithm and how to use the open-source R package FLOWMAPR via its GUI or with text-based commands. This approach can be applied to many dynamic processes, including in vitro stem cell differentiation, in vivo development, oncogenesis, the emergence of drug resistance and cell signaling dynamics. To demonstrate our approach, we perform a step-by-step analysis of a single-cell mass cytometry time course dataset from mouse embryonic stem cells differentiating into the three germ layers: endoderm, mesoderm and ectoderm. In addition, we demonstrate FLOW-MAP analysis of a previously published scRNAseq dataset. Using both synthetic and experimental datasets for comparison, we perform FLOW-MAP analysis side by side with other single-cell analysis methods, to illustrate when it is advantageous to use the FLOW-MAP approach. The protocol takes between 30 min and 1.5 h to complete.
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http://dx.doi.org/10.1038/s41596-019-0246-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897424PMC
February 2020

Sex Differences in the Blood Transcriptome Identify Robust Changes in Immune Cell Proportions with Aging and Influenza Infection.

Cell Rep 2019 11;29(7):1961-1973.e4

Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Medicine, Division of Biomedical Informatics Research, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Sex differences in autoimmunity and infection suggest that a better understanding of molecular sex differences will improve the diagnosis and treatment of immune-related disease. We identified 144 differentially expressed genes, referred to as immune sex expression signature (iSEXS), between human males and females using an integrated multi-cohort analysis of blood transcriptome profiles from six discovery cohorts from five continents with 458 healthy individuals. We validated iSEXS in 11 additional cohorts of 524 peripheral blood samples. When we separated iSEXS into genes located on sex chromosomes (XY-iSEXS) or autosomes (autosomal-iSEXS), both modules distinguished males and females. iSEXS reflects sex differences in immune cell proportions, with female-associated genes showing higher expression by CD4 T cells and male-associated genes showing higher expression by myeloid cells. Autosomal-iSEXS detected an increase in monocytes with age in females, reflected sex-differential immune cell dynamics during influenza infection, and predicted antibody response in males, but not females.
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http://dx.doi.org/10.1016/j.celrep.2019.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856718PMC
November 2019

Pursuing Human-Relevant Gut Microbiota-Immune Interactions.

Immunity 2019 08;51(2):225-239

Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA; Center for Human Microbiome Studies, Stanford University, Stanford, CA, USA. Electronic address:

The gut microbiota is a complex and plastic network of diverse organisms intricately connected with human physiology. Recent advances in profiling approaches of both the microbiota and the immune system now enable a deeper exploration of immunity-microbiota connections. An important next step is to elucidate a human-relevant "map" of microbial-immune wiring while focusing on animal studies to probe a prioritized subset of interactions. Here, we provide an overview of this field's current status and discuss two approaches for establishing priorities for detailed investigation: (1) longitudinal intervention studies in humans probing the dynamics of both the microbiota and the immune system and (2) the study of traditional populations to assess lost features of human microbial identity whose absence may be contributing to the rise of immunological disorders. These human-centered approaches offer a judicious path forward to understand the impact of the microbiota in immune development and function.
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http://dx.doi.org/10.1016/j.immuni.2019.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205593PMC
August 2019

Gut microbiome transition across a lifestyle gradient in Himalaya.

PLoS Biol 2018 11 15;16(11):e2005396. Epub 2018 Nov 15.

Department of Microbiology and Immunology, Stanford University, Stanford, California, United States of America.

The composition of the gut microbiome in industrialized populations differs from those living traditional lifestyles. However, it has been difficult to separate the contributions of human genetic and geographic factors from lifestyle. Whether shifts away from the foraging lifestyle that characterize much of humanity's past influence the gut microbiome, and to what degree, remains unclear. Here, we characterize the stool bacterial composition of four Himalayan populations to investigate how the gut community changes in response to shifts in traditional human lifestyles. These groups led seminomadic hunting-gathering lifestyles until transitioning to varying levels of agricultural dependence upon farming. The Tharu began farming 250-300 years ago, the Raute and Raji transitioned 30-40 years ago, and the Chepang retain many aspects of a foraging lifestyle. We assess the contributions of dietary and environmental factors on their gut-associated microbes and find that differences in the lifestyles of Himalayan foragers and farmers are strongly correlated with microbial community variation. Furthermore, the gut microbiomes of all four traditional Himalayan populations are distinct from that of the Americans, indicating that industrialization may further exacerbate differences in the gut community. The Chepang foragers harbor an elevated abundance of taxa associated with foragers around the world. Conversely, the gut microbiomes of the populations that have transitioned to farming are more similar to those of Americans, with agricultural dependence and several associated lifestyle and environmental factors correlating with the extent of microbiome divergence from the foraging population. The gut microbiomes of Raute and Raji reveal an intermediate state between the Chepang and Tharu, indicating that divergence from a stereotypical foraging microbiome can occur within a single generation. Our results also show that environmental factors such as drinking water source and solid cooking fuel are significantly associated with the gut microbiome. Despite the pronounced differences in gut bacterial composition across populations, we found little differences in alpha diversity across lifestyles. These findings in genetically similar populations living in the same geographical region establish the key role of lifestyle in determining human gut microbiome composition and point to the next challenging steps of determining how large-scale gut microbiome reconfiguration impacts human biology.
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http://dx.doi.org/10.1371/journal.pbio.2005396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237292PMC
November 2018

Links between environment, diet, and the hunter-gatherer microbiome.

Gut Microbes 2019 17;10(2):216-227. Epub 2018 Aug 17.

a Department of Microbiology and Immunology , Stanford University School of Medicine , Stanford , CA , USA.

The study of traditional populations provides a view of human-associated microbes unperturbed by industrialization, as well as a window into the microbiota that co-evolved with humans. Here we discuss our recent work characterizing the microbiota from the Hadza hunter-gatherers of Tanzania. We found seasonal shifts in bacterial taxa, diversity, and carbohydrate utilization by the microbiota. When compared to the microbiota composition from other populations around the world, the Hadza microbiota shares bacterial families with other traditional societies that are rare or absent from microbiotas of industrialized nations. We present additional observations from the Hadza microbiota and their lifestyle and environment, including microbes detected on hands, water, and animal sources, how the microbiota varies with sex and age, and the short-term effects of introducing agricultural products into the diet. In the context of our previously published findings and of these additional observations, we discuss a path forward for future work.
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http://dx.doi.org/10.1080/19490976.2018.1494103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546328PMC
August 2019

GateFinder: projection-based gating strategy optimization for flow and mass cytometry.

Bioinformatics 2018 12;34(23):4131-4133

Baxter Laboratory in Stem Cell Biology.

Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging.

Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology.

Availability And Implementation: The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247943PMC
December 2018

Multicenter Systems Analysis of Human Blood Reveals Immature Neutrophils in Males and During Pregnancy.

J Immunol 2017 03 8;198(6):2479-2488. Epub 2017 Feb 8.

Division of Translational Medicine, Department of Systems Biology, Sidra Medical and Research Center, 26999 Doha, Qatar;

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.
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http://dx.doi.org/10.4049/jimmunol.1601855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337813PMC
March 2017

Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states.

Nat Med 2017 Feb 16;23(2):174-184. Epub 2017 Jan 16.

CIRID, UMR CNRS 5164, Université Bordeaux 2, Bordeaux Cedex, France.

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-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.
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http://dx.doi.org/10.1038/nm.4267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320935PMC
February 2017

Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in Mycobacterium tuberculosis.

ACS Infect Dis 2016 11 15;2(11):800-806. Epub 2016 Aug 15.

Department of Nutritional Science and Toxicology, University of California, Berkeley , 127 Morgan Hall, Berkeley, California 94720, United States.

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in M. tuberculosis. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis.
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http://dx.doi.org/10.1021/acsinfecdis.6b00106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505729PMC
November 2016

Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy.

J Immunol 2016 12 28;197(11):4482-4492. Epub 2016 Oct 28.

Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine, Stanford, CA 94305;

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 TbetCD4 T cells, CD8 T cells, B cells, and CD56CD16 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.
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http://dx.doi.org/10.4049/jimmunol.1601195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125527PMC
December 2016

In Reply.

Anesthesiology 2016 06;124(6):1414-5

Stanford University School of Medicine, Stanford, California (M.S.A.).

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http://dx.doi.org/10.1097/ALN.0000000000001091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914362PMC
June 2016

Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery.

Anesthesiology 2015 Dec;123(6):1241-55

From the Baxter Laboratory in Stem Cell Biology, Stanford University, Stanford, California (G.K.F., B.G., N.A., G.P.N.); Department of Microbiology and Immunology, Stanford University, Stanford, California (G.K.F., G.P.N.); and Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University School of Medicine, Stanford, California (B.G., E.A.G., M.T., M.S.A.).

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.
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http://dx.doi.org/10.1097/ALN.0000000000000887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681408PMC
December 2015

The effect of microbial colonization on the host proteome varies by gastrointestinal location.

ISME J 2016 May 17;10(5):1170-81. Epub 2015 Nov 17.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.

Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.
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http://dx.doi.org/10.1038/ismej.2015.187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029216PMC
May 2016

Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth.

Cytometry A 2015 Sep 17;87(9):817-29. Epub 2015 Jul 17.

Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University, School of Medicine, Stanford, California, 94305.

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.
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http://dx.doi.org/10.1002/cyto.a.22720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758855PMC
September 2015

IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system.

Science 2015 Jul;349(6244):1259425

Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA. Program in Immunology, Stanford University, Stanford, CA 94305, USA.

Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.
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http://dx.doi.org/10.1126/science.1259425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537647PMC
July 2015

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.

Cytometry A 2014 Dec 1;85(12):1011-9. Epub 2014 Oct 1.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California; Divisions of Hematology and Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, California.

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.
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http://dx.doi.org/10.1002/cyto.a.22573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361015PMC
December 2014

Clinical recovery from surgery correlates with single-cell immune signatures.

Sci Transl Med 2014 Sep;6(255):255ra131

Baxter Laboratory in Stem Cell Biology, Stanford University, Stanford, CA 94305, USA. Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.

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.
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http://dx.doi.org/10.1126/scitranslmed.3009701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334126PMC
September 2014