Holden T Maecker

Stanford Medicine, Stanford, California, United States

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Publications (113)638.79 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
  • Serena Chang, Holbrook Kohrt, Holden T Maecker
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    ABSTRACT: A new era of cancer immunotherapy has brought not only successful cancer vaccines but also immunomodulators, such as those that target checkpoint blockade in order to induce endogenous host immune responses. However, the immune system of cancer patients can be compromised through multiple means, including immune suppression by the tumor and by prior therapies such as chemotherapy and radiation. Therefore, a comprehensive means of assessing patient immunocompetence would seem helpful for determining whether patients are ready to benefit from immunotherapy, and perhaps even which immunotherapy might be most appropriate for them. Unfortunately, there are no standardized tests for immune competence, nor is there agreement on what to measure and what will be predictive of outcome. In this review, we will discuss the technologies and assays that might be most useful for this purpose. We argue for a comprehensive approach that should maximize the chances of developing predictive biomarkers for eventual clinical use.
    Cancer Immunology and Immunotherapy 02/2014; · 3.64 Impact Factor
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    ABSTRACT: Background:Re-exposure to viruses is assumed to strengthen humoral and cellular immunity via the secondary immune response. We studied the effects of frequent exposure to viral infectious challenges on immunity. Furthermore, we assessed whether repetitive re-exposures to varicella-zoster virus (VZV) elicited persistently high immune responses.Methods:Blood samples from 11 pediatricians and matched controls were assessed at 3 time points and 1 time point, respectively. Besides the assessment of general immunity by means of T-cell subset percentages, antibody titers and IFN-gamma/IL-2-producing T-cell percentages against adenovirus type 5 (AdV-5), cytomegalovirus (CMV), Tetanus toxin (TT) and VZV were determined.Results:Pediatricians had lower levels of circulating CD4+ naïve T-cells and showed boosting of CD8+ effector memory T-cells. Although no effect on humoral immunity was seen, repetitive re-exposure to VZV induced persistently higher percentages of IFN-gamma positive T-cells against all VZV antigens tested (VZV gE, VZV IE62, VZV IE63) when compared to controls. T-cells directed against latency-associated VZV IE63 benefitted the most from natural exogenous boosting. Although no differences in cellular or humoral immunity were found between pediatricians and controls for AdV-5 or TT, we did find larger immune responses against CMV antigens in pediatricians.Conclusions:Despite the high infectious burden we detected a robust and diverse immune system in pediatricians. Repetitive re-exposure to VZV has been shown to induce a stable increased level of VZV-specific cellular, but not humoral immunity. Based on our observations, VZV IE63 could be considered as one of the candidates for a zoster vaccine.
    Clinical and vaccine Immunology: CVI 01/2014; · 2.60 Impact Factor
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    ABSTRACT: Recently, a framework for structured reporting of results from T cell assays (MIATA: Minimal Information About T cell Assays) has been added to field-wide ongoing efforts to improve the quality of immune monitoring (van der Burg et al., 2011; Britten et al., 2012). Immune monitoring plays an increasing role in clinical immunology and development of novel therapeutics as well as in basic immunological research. Independent of the application, MIATA aims to increase transparency and stimulate efforts towards higher quality in the assay conduct.
    Journal of immunological methods 01/2014; · 2.35 Impact Factor
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    ABSTRACT: Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or “split” viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors—specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus “splitting” inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.
    Vaccine. 01/2014;
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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. · 3.53 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

Publication Stats

4k Citations
638.79 Total Impact Points

Institutions

  • 1997–2014
    • Stanford Medicine
      • • Department of Microbiology and Immunology
      • • Division of Oncology
      • • Department of Medicine
      Stanford, California, United States
    • Stanford University
      • • Department of Medicine
      • • Department of Microbiology and Immunology
      • • Institute for Immunity, Transplantation and Infection
      • • Department of Pediatrics
      Palo Alto, California, United States
  • 2013
    • Children's Hospital Los Angeles
      Los Angeles, California, 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
  • 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