Holden T Maecker

Children's Hospital Los Angeles, Los Angeles, California, United States

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Publications (109)623.32 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Multiplexed fluorescence or electrochemiluminescence immunoassays of soluble cytokines are commonly performed in the context of human serum or plasma, to look for disease biomarkers and to monitor the immune system in a simple and minimally invasive way. These assays provide challenges due to the complexities of the matrix (serum or plasma) and the presence of many cytokines near the limit of detection of the assay. Here, we compare the readout of matched serum and plasma samples, which are generally correlated. However, a subset of cytokines usually have higher levels in serum, and the non-specific background is significantly increased in serum versus plasma. Presumably as a result of this non-specific background, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum. We further show, through spike recovery experiments, that both serum and plasma inhibit the readout of many cytokines, with some variability between donors, but with serum causing greater inhibition than plasma in many cases. Standard diluents from different vendors can partially reverse this inhibition to varying degrees. Dilution of samples can also partly overcome the inhibitory effect of the matrix. We also show that dilution is nonlinear and differentially affects various cytokines. Together, these data argue that (1) plasma is a more sensitive matrix for detecting changes in certain low-abundance cytokines; (2) calculation of concentrations in serum or plasma matrices is inherently inaccurate; and (3) dilution of samples should not be assumed to be linear, i.e., all comparisons need to be made among similarly diluted samples.
    Immunologic Research 02/2014; · 2.96 Impact Factor
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    ABSTRACT: In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals. Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African-Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed. Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4+ (p = 0.04) and CD8+ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels. A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.
    PLoS ONE 01/2014; 9(4):e94500. · 3.73 Impact Factor
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    ABSTRACT: We mapped the cytokine response to hematopoietic stem cell transplantation (HSCT) by assaying fifty-one cytokines and chemokines (Luminex multiplex cytokine kits, Panomics/Affymetrix, Santa Clara, CA) each week for one hundred days in fifty-one children receiving allogeneic (n=44) or autologous HSCT (n=7). Assay values were reported as mean fluorescence intensity (MFI). Log transformation converted MFI to clinically relevant measures (i.e., picograms/mL). We searched for potential markers of transplant complications by employing mixed treatment by subjects ANOVA (analysis of variance). Global cytokine secretion in HSCT recipients was significantly lower than in concurrent control patients (n=11). Coincident with the nadir in white blood cell count, the concentration of many cytokines declined further by the second and third week: CD40, FGF-β, ICAM1, IL-1α, IL-1β, IL-2, IL-5, IL-4, IL-7, IL-12P40, IL-12P70, IL-13, IL-15, IL-17, interferon, IP-10, MCP3, MIG, MIP1β, NGF, PAI1, PDGFBB, RANTES, resistin, sFAS ligand, TNF-α, TGF-α, TGF-β, TRAIL, VCAM1. All of these analytes (except MIG) subsequently rebounded by week 4 (coincident with engraftment and recovery of the white blood cell count), but often still remained well below the control levels. Concurrent with the collective nadir of multiple cytokines, MCP-1, GRO-α and leptin surged during weeks 2-4. High levels of leptin persisted throughout the one hundred post-transplant days. Also during weeks 2-4, hepatocyte growth factor (HGF) and interleukin-6 (IL-6) surged in children with complications, but not in those without complications. The peak in HGF was more pronounced in veno-occlusive disease (VOD). HGF and IL-6 secretion rose at least two weeks prior to the clinical diagnosis of VOD or graft-vs-host disease (GVHD). From week 4 onwards in all groups, the MFI of the cytokine resistin increased to 5-15 times above concurrent control. Hepatocyte growth factor has now emerged in three or more biomarker discovery efforts for GVHD (and in our population, for VOD as well). Hepatocyte growth factor (with or without IL-6) should be investigated as a potential predictive biomarker of VOD or GVHD. Alternatively, the hyper-inflammatory 'signature' provided by a multi-cytokine assay may be predictive.
    Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 12/2013; · 3.15 Impact Factor
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    ABSTRACT: Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.
    Science translational medicine 10/2013; 5(208):208ra145. · 10.76 Impact Factor
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    ABSTRACT: Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling. © 2013 International Society for Advancement of Cytometry.
    Cytometry Part A 06/2013; · 3.71 Impact Factor
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    ABSTRACT: BACKGROUND: Chronic fatigue syndrome (CFS) is a debilitating disorder characterized by persistent fatigue that is not alleviated by rest. The lack of a clearly identified underlying mechanism has hindered the development of effective treatments. Studies have demonstrated elevated levels of inflammatory factors in patients with CFS, but findings are contradictory across studies and no biomarkers have been consistently supported. Single time-point approaches potentially overlook important features of CFS, such as fluctuations in fatigue severity. We have observed that individuals with CFS demonstrate significant day-to-day variability in their fatigue severity. METHODS: Therefore, to complement previous studies, we implemented a novel longitudinal study design to investigate the role of cytokines in CFS pathophysiology. Ten women meeting the Fukuda diagnostic criteria for CFS and ten healthy age- and body mass index (BMI)-matched women underwent 25 consecutive days of blood draws and self-reporting of symptom severity. A 51-plex cytokine panel via Luminex was performed for each of the 500 serum samples collected. Our primary hypothesis was that daily fatigue severity would be significantly correlated with the inflammatory adipokine leptin, in the women with CFS and not in the healthy control women. As a post-hoc analysis, a machine learning algorithm using all 51 cytokines was implemented to determine whether immune factors could distinguish high from low fatigue days. RESULTS: Self-reported fatigue severity was significantly correlated with leptin levels in six of the participants with CFS and one healthy control, supporting our primary hypothesis. The machine learning algorithm distinguished high from low fatigue days in the CFS group with 78.3% accuracy. CONCLUSIONS: Our results support the role of cytokines in the pathophysiology of CFS.
    Journal of Translational Medicine 04/2013; 11(1):93. · 3.46 Impact Factor
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    ABSTRACT: Despite the importance of the immune system in many diseases, there are currently no objective benchmarks of immunological health. In an effort to identifying such markers, we used influenza vaccination in 30 young (20-30 years) and 59 older subjects (60 to >89 years) as models for strong and weak immune responses, respectively, and assayed their serological responses to influenza strains as well as a wide variety of other parameters, including gene expression, antibodies to hemagglutinin peptides, serum cytokines, cell subset phenotypes and in vitro cytokine stimulation. Using machine learning, we identified nine variables that predict the antibody response with 84% accuracy. Two of these variables are involved in apoptosis, which positively associated with the response to vaccination and was confirmed to be a contributor to vaccine responsiveness in mice. The identification of these biomarkers provides new insights into what immune features may be most important for immune health.
    Molecular Systems Biology 01/2013; 9:659. · 11.34 Impact Factor
  • Nature Reviews Rheumatology 10/2012; 8(10):562. · 9.75 Impact Factor
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    ABSTRACT: Recent publications have suggested that altered proportions of functional CD4 T-cell subsets correlate with active pulmonary TB. Also, CD27-expression on tuberculin-activated IFN-γ(+) CD4 T-cells is known to differ significantly between patients with active pulmonary TB and healthy TB-unexposed BCG vaccinees. Here, we explore links between CD4 T-cell phenotype, multiple functional subsets, and control of TB. We examined ex-vivo overnight tuberculin activated CD4 T-cells in regards to CD27-expression and the activation markers, CD154 upregulation, IFN-γ, TNF-α, IL-2, and degranulation in 44 individuals, including cases of clinically active pulmonary TB, and hospital staff with prolonged TB exposure, some of whom had latent TB. Active pulmonary TB generally showed an excess of TNF-α(+) subsets over IFN-γ(+) subsets, paralleled by decreased CD27 expression on activated IFN-γ(+) or CD154(+) CD4 T-cells. The single subset distinguishing best between active pulmonary TB and high TB exposure was CD154(+) /TNF-α(+) / IFN-γ(-) /IL-2(-) /degranulation(-) (AUROC 0.90). The ratio between the frequencies of TNF-α(+) /IFN-γ(+) CD4 T-cells was an effective alternative parameter (AUROC 0.87). Functional subsets and phenotype of tuberculin induced CD4 T-cells differ between stages of TB infection. Predominance of TNF-α(+) CD4 T-cells in active infection suggests an increased effort of the immune system to contain disease. © 2012 International Clinical Cytometry Society.
    Cytometry Part B Clinical Cytometry 09/2012; 82(6):360-8. · 2.23 Impact Factor
  • Arthritis Research & Therapy 09/2012; 14(3). · 4.30 Impact Factor
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    Immunity 07/2012; 37(1):1-2. · 19.80 Impact Factor
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    ABSTRACT: Systems-level approaches are increasingly common in both murine and human translational studies. These approaches employ multiple high information content assays. As a result, there is a need for tools to integrate heterogeneous types of laboratory and clinical/demographic data, and to allow the exploration of that data by aggregating and/or segregating results based on particular variables (e.g., mean cytokine levels by age and gender). Here we describe the application of standard data warehousing tools to create a novel environment for user-driven upload, integration, and exploration of heterogeneous data. The system presented here currently supports flow cytometry and immunoassays performed in the Stanford Human Immune Monitoring Center, but could be applied more generally. Users upload assay results contained in platform-specific spreadsheets of a defined format, and clinical and demographic data in spreadsheets of flexible format. Users then map sample IDs to connect the assay results with the metadata. An OLAP (on-line analytical processing) data exploration interface allows filtering and display of various dimensions (e.g., Luminex analytes in rows, treatment group in columns, filtered on a particular study). Statistics such as mean, median, and N can be displayed. The views can be expanded or contracted to aggregate or segregate data at various levels. Individual-level data is accessible with a single click. The result is a user-driven system that permits data integration and exploration in a variety of settings. We show how the system can be used to find gender-specific differences in serum cytokine levels, and compare them across experiments and assay types. We have used the tools and techniques of data warehousing, including open-source business intelligence software, to support investigator-driven data integration and mining of diverse immunological data.
    Journal of Translational Medicine 03/2012; 10:62. · 3.46 Impact Factor
  • Holden T Maecker, J Philip McCoy, Robert Nussenblatt
    Nature Reviews Immunology 01/2012; 12(5):396. · 32.25 Impact Factor
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    ABSTRACT: Rheumatologists see patients with a range of autoimmune diseases. Phenotyping these diseases for diagnosis, prognosis and selection of therapies is an ever increasing problem. Advances in multiplexed assay technology at the gene, protein, and cellular level have enabled the identification of 'actionable biomarkers'; that is, biological metrics that can inform clinical practice. Not only will such biomarkers yield insight into the development, remission, and exacerbation of a disease, they will undoubtedly improve diagnostic sensitivity and accuracy of classification, and ultimately guide treatment. This Review provides an introduction to these powerful technologies that could promote the identification of actionable biomarkers, including mass cytometry, protein arrays, and immunoglobulin and T-cell receptor high-throughput sequencing. In our opinion, these technologies should become part of routine clinical practice for the management of autoimmune diseases. The use of analytical tools to deconvolve the data obtained from use of these technologies is also presented here. These analyses are revealing a more comprehensive and interconnected view of the immune system than ever before and should have an important role in directing future treatment approaches for autoimmune diseases.
    Nature Reviews Rheumatology 01/2012; 8(6):317-28. · 9.75 Impact Factor
  • Michael D Leipold, Holden T Maecker
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    ABSTRACT: In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.(1) Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.(2-5) Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.(6-7) Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells. Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.
    Journal of Visualized Experiments 01/2012;
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    Holden T Maecker, J Philip McCoy, Robert Nussenblatt
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    ABSTRACT: The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.
    Nature Reviews Immunology 01/2012; 12(3):191-200. · 32.25 Impact Factor
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    ABSTRACT: Although BCR-ABL+ stem cells in chronic myeloid leukemia (CML) resist elimination by targeted pharmacotherapy in most patients, immunological graft-versus-leukemia effects can cure the disease. Besides cytotoxic T cells, natural killer (NK) cells may have a role in immune control of CML. Here, we explored the functionality of NK cells in CML patients and in a transgenic inducible BCR-ABL mouse model. Compared with controls, NK-cell proportions among lymphocytes were decreased at diagnosis of CML and did not recover during imatinib-induced remission for 10-34 months. Functional experiments revealed limited in vitro expansion of NK cells from CML patients and a reduced degranulation response to K562 target cells both at diagnosis and during imatinib therapy. Consistent with the results in human CML, relative numbers of NK1.1+ NK cells were reduced following induction of BCR-ABL expression in mice, and the defects persisted after BCR-ABL reversion. Moreover, target-induced degranulation by expanded BCR-ABL+ NK cells was compromised. We conclude that CML is associated with quantitative and functional defects within the NK-cell compartment, which is reproduced by induced BCR-ABL expression in mice. Further work will aim at identifying the mechanisms of NK-cell deficiency in CML and at developing strategies to exploit NK cells for immunotherapy.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2011; 26(3):465-74. · 10.16 Impact Factor
  • The Journal of Infectious Diseases 07/2011; 204(2):327-8. · 5.85 Impact Factor

Publication Stats

4k Citations
1k Downloads
623.32 Total Impact Points

Institutions

  • 2013
    • Children's Hospital Los Angeles
      Los Angeles, California, United States
  • 1997–2013
    • Stanford University
      • • Department of Medicine
      • • Department of Microbiology and Immunology
      • • Institute for Immunity, Transplantation and Infection
      • • Department of Pediatrics
      Palo Alto, CA, United States
  • 2010–2012
    • Johannes Gutenberg-Universität Mainz
      • III. Department of Medicine
      Mayence, Rheinland-Pfalz, Germany
  • 2007–2012
    • Charité Universitätsmedizin Berlin
      • Institute of Medical Immunology
      Berlin, Land Berlin, Germany
  • 1997–2012
    • Stanford Medicine
      • Department of Medicine
      Stanford, California, United States
  • 2011
    • University of Pittsburgh
      • School of Medicine
      Pittsburgh, PA, United States
  • 2003–2009
    • Becton, Dickinson and Company (BD)
      • BD Biosciences
      Franklin Lakes, New Jersey, United States
  • 2006
    • University of Washington Seattle
      • Center for Translational Medicine in Women's Health
      Seattle, WA, United States
  • 2004
    • Oregon Health and Science University
      • Vaccine and Gene Therapy Institute
      Portland, OR, United States