Academic Appointments

Administrative Appointments

  • Director, Human Immune Monitoring Center (2009 - Present)

Professional Education

  • PhD, Stanford University, Cancer Biology (1988)
  • BS, Purdue University, Microbiology (1984)

Community and International Work

  • Human Immunophenotyping Consortium


    Standardization of human immune monitoring

    Partnering Organization(s)




    Ongoing Project


    Opportunities for Student Involvement


Current Research and Scholarly Interests

Our lab’s research focuses on cellular immune responses to pathogens and cancer, and the correlation of those responses with protection from clinical disease. We use systems-level immune monitoring assays, and currently have projects focused on cytomegalovirus (CMV), EBV, HIV, and cancer. These are all settings where cellular immunity is important, and thus we wish to map the detailed phenotypes and functions of the immune cells involved. Correlating these phenotypes and functions with disease outcome or therapeutic responsiveness would enable better vaccine development and monitoring of individuals undergoing immunotherapy.

Clinical Trials

  • Effect of Celecoxib on Perioperative Inflammatory Response in Colon Cancer Not Recruiting

    The proposed study aims to investigate how the administration of a drug known to reduce inflammation in humans, Celecoxib, will effect the peri-operative inflammatory response of a patient undergoing primary tumor resection surgery for colon cancer. The proposed project is an exploratory study, and will use data from blood samples and tumor samples to attempt to elucidate the immune and inflammatory response in colon cancer patients undergoing primary resection of their tumors.

    Stanford is currently not accepting patients for this trial. For more information, please contact Julia McNeal, (650) 723 - 9433.

    View full details

2013-14 Courses

Postdoctoral Advisees

Journal Articles

  • A harmonized approach to intracellular cytokine staining gating: Results from an international multiconsortia proficiency panel conducted by the Cancer Immunotherapy Consortium (CIC/CRI). Cytometry. Part A : the journal of the International Society for Analytical Cytology McNeil, L. K., Price, L., Britten, C. M., Jaimes, M., Maecker, H., Odunsi, K., Matsuzaki, J., Staats, J. S., Thorpe, J., Yuan, J., Janetzki, S. 2013; 83 (8): 728-738


    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.

    View details for DOI 10.1002/cyto.a.22319

    View details for PubMedID 23788464

  • Experimental Pain and Opioid Analgesia in Volunteers at High Risk for Obstructive Sleep Apnea PLOS ONE Doufas, A. G., Tian, L., Padrez, K. A., Suwanprathes, P., Cardell, J. A., Maecker, H. T., Panousis, P. 2013; 8 (1)


    Obstructive sleep apnea (OSA) is characterized by recurrent nocturnal hypoxia and sleep disruption. Sleep fragmentation caused hyperalgesia in volunteers, while nocturnal hypoxemia enhanced morphine analgesic potency in children with OSA. This evidence directly relates to surgical OSA patients who are at risk for airway compromise due to postoperative use of opioids. Using accepted experimental pain models, we characterized pain processing and opioid analgesia in male volunteers recruited based on their risk for OSA.After approval from the Intitutional Review Board and informed consent, we assessed heat and cold pain thresholds and tolerances in volunteers after overnight polysomnography (PSG). Three pro-inflammatory and 3 hypoxia markers were determined in the serum. Pain tests were performed at baseline, placebo, and two effect site concentrations of remifentanil (1 and 2 µg/ml), an ?-opioid agonist. Linear mixed effects regression models were employed to evaluate the association of 3 PSG descriptors [wake after sleep onset, number of sleep stage shifts, and lowest oxyhemoglobin saturation (SaO(2)) during sleep] and all serum markers with pain thresholds and tolerances at baseline, as well as their changes under remifentanil.Forty-three volunteers (12 normal and 31 with a PSG-based diagnosis of OSA) were included in the analysis. The lower nadir SaO(2) and higher insulin growth factor binding protein-1 (IGFBP-1) were associated with higher analgesic sensitivity to remifentanil (SaO(2), P = 0.0440; IGFBP-1, P = 0.0013). Other pro-inflammatory mediators like interleukin-1? and tumor necrosis factor-? (TNF-?) were associated with an enhanced sensitivity to the opioid analgesic effect (IL-1?, P = 0.0218; TNF-?, P = 0.0276).Nocturnal hypoxemia in subjects at high risk for OSA was associated with an increased potency of opioid analgesia. A serum hypoxia marker (IGFBP-1) was associated with hypoalgesia and increased potency to opioid analgesia; other pro-inflammatory mediators also predicted an enhanced opioid NCT00672737.

    View details for DOI 10.1371/journal.pone.0054807

    View details for Web of Science ID 000315483200027

    View details for PubMedID 23382975

  • Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Molecular systems biology Furman, D., Jojic, V., Kidd, B., Shen-Orr, S., Price, J., Jarrell, J., Tse, T., Huang, H., Lund, P., Maecker, H. T., Utz, P. J., Dekker, C. L., Koller, D., Davis, M. M. 2013; 9: 659-?


    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.

    View details for DOI 10.1038/msb.2013.15

    View details for PubMedID 23591775

  • Improved accuracy and reproducibility of enumeration of platelet-monocyte complexes through use of doublet-discriminator strategy CYTOMETRY PART B-CLINICAL CYTOMETRY Streitz, M., Fuhrmann, S., Thomas, D., Cheek, E., Nomura, L., Maecker, H., Martus, P., Aghaeepour, N., Brinkman, R. R., Volk, H., Kern, F. 2012; 82B (6): 360-368


    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.

    View details for DOI 10.1002/cyto.b.21040

    View details for Web of Science ID 000310386300003

  • T Cell Assays and MIATA: The Essential Minimum for Maximum Impact IMMUNITY Britten, C. M., Janetzki, S., Butterfield, L. H., Ferrari, G., Gouttefangeas, C., Huber, C., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J., O'Donnell-Tormey, J., Odunsi, K., Old, L. J., Ottenhoff, T. H., Ottensmeier, C., Pawelec, G., Roederer, M., Roep, B. O., Romero, P., van der Burg, S. H., Walter, S., Hoos, A., DAVIS, M. M. 2012; 37 (1): 1-2

    View details for DOI 10.1016/j.immuni.2012.07.010

    View details for Web of Science ID 000307133200001

    View details for PubMedID 22840835

  • New tools for classification and monitoring of autoimmune diseases NATURE REVIEWS RHEUMATOLOGY Maecker, H. T., Lindstrom, T. M., Robinson, W. H., Utz, P. J., Hale, M., Boyd, S. D., Shen-Orr, S. S., Fathman, C. G. 2012; 8 (6): 317-328


    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.

    View details for DOI 10.1038/nrrheum.2012.66

    View details for Web of Science ID 000304719600005

    View details for PubMedID 22647780

  • The Stanford Data Miner: a novel approach for integrating and exploring heterogeneous immunological data JOURNAL OF TRANSLATIONAL MEDICINE Siebert, J. C., Munsil, W., Rosenberg-Hasson, Y., Davis, M. M., Maecker, H. T. 2012; 10


    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.

    View details for DOI 10.1186/1479-5876-10-62

    View details for Web of Science ID 000304554800001

    View details for PubMedID 22452993

  • NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR-ABL-positive mice LEUKEMIA Chen, C., Koschmieder, S., KERSTIENS, L., Schemionek, M., Altvater, B., Pscherer, S., Gerss, J., Maecker, H. T., Berdel, W. E., Juergens, H., Lee, P. P., Rossig, C. 2012; 26 (3): 465-474


    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.

    View details for DOI 10.1038/leu.2011.239

    View details for Web of Science ID 000301290300012

    View details for PubMedID 21904381

  • Standardizing immunophenotyping for the Human Immunology Project NATURE REVIEWS IMMUNOLOGY Maecker, H. T., McCoy, J. P., Nussenblatt, R. 2012; 12 (3): 191-200


    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.

    View details for DOI 10.1038/nri3158

    View details for Web of Science ID 000300790600013

    View details for PubMedID 22343568

  • Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer. Journal of visualized experiments : JoVE Leipold, M. D., Maecker, H. T. 2012: e4398-?


    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. Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes. 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. 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.

    View details for DOI 10.3791/4398

    View details for PubMedID 23149654

  • Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers CLINICAL CANCER RESEARCH Butterfield, L. H., Palucka, A. K., Britten, C. M., Dhodapkar, M. V., Hakansson, L., Janetzki, S., Kawakami, Y., Kleen, T., Lee, P. P., Maccalli, C., Maecker, H. T., Maino, V. C., Maio, M., Malyguine, A., Masucci, G., Pawelec, G., Potter, D. M., Rivoltini, L., Salazar, L. G., Schendel, D. J., Slingluff, C. L., Song, W., Stroncek, D. F., Tahara, H., Thurin, M., Trinchieri, G., van der Burg, S. H., Whiteside, T. L., Wigginton, J. M., Marincola, F., Khleif, S., Fox, B. A., Disis, M. L. 2011; 17 (10): 3064-3076


    To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements.The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations.Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.

    View details for DOI 10.1158/1078-0432.CCR-10-2234

    View details for Web of Science ID 000290610000003

    View details for PubMedID 21558394

  • Tuberculin-Specific T Cells Are Reduced in Active Pulmonary Tuberculosis Compared to LTBI or Status Post BCG Vaccination JOURNAL OF INFECTIOUS DISEASES Streitz, M., Fuhrmann, S., Powell, F., Quassem, A., Nomura, L., Maecker, H., Martus, P., Volk, H., Kern, F. 2011; 203 (3): 378-382


    Functional characteristics of tuberculosis (TB)-specific CD4 T cells were studied in clinically active pulmonary TB (n?=?21) and high TB exposure including LTBI (n?=?17). Following tuberculin stimulation, activated CD4 T cells were identified by flow-cytometry (CD154 up-regulation, degranulation, interferon ? [IFN-?], tumor necrosis factor ? [TNF-?], and interleukin 2 [IL-2\ production). Interestingly, CD154 up-regulation accounted for ?80% of activated CD4 T cells in the active TB group but just 40% in the controls, whereas IFN-? accounted for only ?50% of activated cells in each group. The frequencies of CD4 T cells displaying at least 1 activation marker discriminated better between the groups than those displaying degranulation or IFN-? production alone.

    View details for DOI 10.1093/infdis/jiq065

    View details for Web of Science ID 000286611800016

    View details for PubMedID 21186260

  • Quality assurance of intracellular cytokine staining assays: Analysis of multiple rounds of proficiency testing JOURNAL OF IMMUNOLOGICAL METHODS Jaimes, M. C., Maecker, H. T., Yan, M., Maino, V. C., Hanley, M. B., Greer, A., Darden, J. M., D'Souza, M. P. 2011; 363 (2): 143-157


    When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-?, IL-2 and/or TNF-? responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination was used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.

    View details for DOI 10.1016/j.jim.2010.08.004

    View details for Web of Science ID 000287176100006

    View details for PubMedID 20727897

  • Multiparameter intracellular cytokine staining. Methods in molecular biology (Clifton, N.J.) Lovelace, P., Maecker, H. T. 2011; 699: 165-178


    Intracellular cytokine staining (ICS) is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g. effector and memory T-cell compartments. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 8-12 (or more) markers in combination, creating technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor ICS, and present an optimized protocol with variations designed for use with specific markers and sample types.

    View details for DOI 10.1007/978-1-61737-950-5_8

    View details for PubMedID 21116983

  • Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond CANCER IMMUNOLOGY IMMUNOTHERAPY Britten, C. M., Janetzki, S., van der Burg, S. H., Huber, C., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J., O'Donnell-Tormey, J., Odunsi, K., Old, L. J., Pawelec, G., Roep, B. O., Romero, P., Hoos, A., DAVIS, M. M. 2011; 60 (1): 15-22


    Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project.

    View details for DOI 10.1007/s00262-010-0940-z

    View details for Web of Science ID 000286662100002

    View details for PubMedID 21080166

  • A model for harmonizing flow cytometry in clinical trials. Nature immunology Maecker, H. T., McCoy, J. P., Amos, M., Elliott, J., Gaigalas, A., Wang, L., Aranda, R., Banchereau, J., Boshoff, C., Braun, J., Korin, Y., Reed, E., Cho, J., Hafler, D., Davis, M., Fathman, C. G., Robinson, W., Denny, T., Weinhold, K., Desai, B., Diamond, B., Gregersen, P., Di Meglio, P., Nestle, F. O., Peakman, M., Villanova, F., Ferbas, J., Field, E., Kantor, A., Kawabata, T., Komocsar, W., Lotze, M., Nepom, J., Ochs, H., O'Lone, R., Phippard, D., Plevy, S., Rich, S., Roederer, M., Rotrosen, D., Yeh, J. 2010; 11 (11): 975-978


    Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

    View details for DOI 10.1038/ni1110-975

    View details for PubMedID 20959798

  • New technologies for autoimmune disease monitoring CURRENT OPINION IN ENDOCRINOLOGY DIABETES AND OBESITY Maecker, H. T., Nolan, G. P., Fathman, C. G. 2010; 17 (4): 322-328


    This article will review new technologies used to characterize the immune phenotype of cells and serum for potential use in studies of autoimmunity.One area of recent development in studies of immune phenotyping is the contrast between cells of the immune system at rest and following activation. This simply involves comparing these cells at rest and following ligand-induced activation and measuring signaling system activation (phosphoepitope identification) or intracellular cytokine production or activation-induced gene expression. Preliminary data using these techniques have begun to identify signatures of disease (biomarkers) that are only seen when using these activation-induced assays. One of the most exciting new technologies, cytometry by time-of-flight mass spectrometry, couples a flow cytometer to a mass spectrometer, allowing many more parameters to be analyzed per cell, and without spillover between assay reagents, compared to conventional optical flow cytometry (heavy ions, mass, replaces fluorophore readout). Another new technology to analyze soluble proteins, bead-based immunoassays, can simultaneously measure up to 75 soluble analytes in a multiplexed array. Other technologies provide similar innovations in terms of analytical content, throughput, and miniaturization.We believe that new cellular genomic and protein-based technologies can provide key insights into autoimmune disease pathogenesis, progression, and therapy, and that these assays need to be applied in a systematic way to samples from patients with autoimmune diseases.

    View details for DOI 10.1097/MED.0b013e32833ada91

    View details for Web of Science ID 000285063800003

    View details for PubMedID 20531181

  • "MIATA"-Minimal Information about T Cell Assays IMMUNITY Janetzki, S., Britten, C. M., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J., Old, L. J., Romero, P., Hoos, A., Davis, M. M. 2009; 31 (4): 527-528


    Immunotherapy, especially therapeutic vaccination, has a great deal of potential in the treatment of cancer and certain infectious diseases such as HIV (Allison et al., 2006; Fauci et al., 2008; Feldmann and Steinman, 2005). Numerous vaccine candidates have been tested in patients with a variety of tumor types and chronic viral diseases. Often, the best way to assess the clinical potential of these vaccines is to monitor the induced T cell response, and yet there are currently no standards for reporting these results. This letter is an effort to address this problem.

    View details for DOI 10.1016/j.immuni.2009.09.007

    View details for Web of Science ID 000271403900001

    View details for PubMedID 19833080

  • Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium CANCER IMMUNOLOGY IMMUNOTHERAPY Britten, C. M., Janetzki, S., Ben-Porat, L., Clay, T. M., Kalos, M., Maecker, H., Odunsi, K., Pride, M., Old, L., Hoos, A., Romero, P. 2009; 58 (10): 1701-1713


    The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay.Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis.We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions.Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.

    View details for DOI 10.1007/s00262-009-0681-z

    View details for Web of Science ID 000268294400016

    View details for PubMedID 19259668

  • Multiparameter flow cytometry monitoring of T cell responses. Methods in molecular biology (Clifton, N.J.) Maecker, H. T. 2009; 485: 375-391


    HIV vaccine research increasingly uses polychromatic flow cytometry as a tool to monitor T cell responses. The use of this technology allows for the analysis of highly defined subsets of cells with unique phenotypes and functions. Ultimately, such studies may identify surrogate markers of protection from disease progression. However, this powerful technology comes with a number of technical hurdles, and there is a need to standardize the assays and protocols used in clinical trial monitoring. Here an optimized protocol, with variations for specific circumstances, is presented. This protocol covers the analysis of multiple cytokines, cell surface markers, and other functional markers such as perforin, CD107, and CD154. While the protocol can be adapted to various numbers of fluorescence parameters, optimized panels of 8-10 colors are presented.

    View details for DOI 10.1007/978-1-59745-170-3_25

    View details for PubMedID 19020838

  • A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers" JOURNAL OF TRANSLATIONAL MEDICINE Butterfield, L. H., Disis, M. L., Fox, B. A., Lee, P. P., Khleif, S. N., Thurin, M., Trinchieri, G., Wang, E., Wigginton, J., Chaussabel, D., Coukos, G., Dhodapkar, M., Hakansson, L., Janetzki, S., Kleen, T. O., Kirkwood, J. M., Maccalli, C., Maecker, H., Maio, M., Malyguine, A., Masucci, G., Palucka, A. K., Potter, D. M., Ribas, A., Rivoltini, L., Schendel, D., Seliger, B., Selvan, S., Slingluff, C. L., Stroncek, D. F., Streicher, H., Wu, X., Zeskind, B., Zhao, Y., Zocca, M., Zwierzina, H., Marincola, F. M. 2008; 6


    The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document.

    View details for DOI 10.1186/1479-5876-6-81

    View details for Web of Science ID 000263537200001

    View details for PubMedID 19105846

  • Standardization and Optimization of Multiparameter Intracellular Cytokine Staining CYTOMETRY PART A Nomura, L., Maino, V. C., Maecker, H. T. 2008; 73A (11): 984-991


    Intracellular cytokine staining (ICS) is a common method for rapid quantitation of cytokine-producing antigen-specific T cells. T cell production of IFNgamma in particular, and more recently IL-2 as well, is often taken as a measure of vaccine immunogenicity in experimental vaccine trials. As more fluorochromes become available for use in ICS and other applications detecting intracellular markers, the selection of optimal fluorochrome combinations becomes correspondingly more complicated. Additionally, as more sophisticated flow cytometers become available, more attention is being paid to potential result variability from one instrument to another. This review summarizes an oral presentation given at MASIR 2008, January 30-Feb 1, 2008, in La Plagne, France. We focus on issues associated with multiparameter (>four color) flow cytometry, including matching antibody specificities with available fluorochromes and techniques to optimize fluorochrome combinations. We examine issues specific to intracellular staining as well as broader topics such as instrument setup, experimental controls, sample management, and analysis of multiparameter data sets. Particular emphasis is placed on the use of lyophilized cells, antibodies, beads, peptides, etc. (collectively known as "lyoplates"), which can decrease experiment-to-experiment variability as well as processing time. Most clinical trials compile results from multiple testing sites, using data that was acquired on-site in each location. We present data from two different ongoing multi-laboratory standardization studies, one involving 15 laboratories and one involving nine. We identify issues of variability and, where possible, offer solutions.

    View details for DOI 10.1002/cyto.a.20602

    View details for Web of Science ID 000260622300003

  • Development and dynamics of robust T-cell responses to CML under imatinib treatment patients BLOOD Chen, C. I., Maecker, H. T., Lee, P. P. 2008; 111 (11): 5342-5349


    Novel molecular targeted therapies, such as imatinib for chronic myelogenous leukemia (CML), represent the first agents that inhibit cancer cells more than other dividing cells, such as immune cells. We hypothesize that imatinib may create a window in which the immune response is partially restored while apoptotic leukemic cells are present, thus rendering leukemic cells immunogenic as patients enter remission. To detect and quantify antileukemia immune responses in an antigen-unbiased way, we used cryopreserved autologous pretreatment blood samples (representing predominantly leukemic cells) as stimulators to detect antileukemia T-cell responses in CML patients in remission on imatinib. We studied patients over time to address the dynamics of such responses. Our data show that antileukemia T-cell responses develop in the majority of CML patients (9 of 14) in remission and that CD4(+) T cells producing tumor necrosis factor-alpha (median 17.6%) represent the major response over interferon-gamma. This confirms the immune system's ability to respond to leukemia under certain conditions. Such responses may be further amplified as a potential therapy that synergizes with imatinib for improved control of CML.

    View details for DOI 10.1182/blood-2007-12-128397

    View details for Web of Science ID 000256336500016

    View details for PubMedID 18326818

  • An analytical workflow for investigating cytokine profiles CYTOMETRY PART A Siebert, J. C., Inokurna, M., Waid, D. M., Pennock, N. D., Vaitaitis, G. M., Disis, M. L., Dunne, J. R., Wagner, D. H., Maecker, H. T. 2008; 73A (4): 289-298


    Understanding cytokine profiles of disease states has provided researchers with great insight into immunologic signaling associated with disease onset and progression, affording opportunities for advancement in diagnostics and therapeutic intervention. Multiparameter flow cytometric assays support identification of specific cytokine secreting subpopulations. Bead-based assays provide simultaneous measurement for the production of ever-growing numbers of cytokines. These technologies demand appropriate analytical techniques to extract relevant information efficiently. We illustrate the power of an analytical workflow to reveal significant alterations in T-cell cytokine expression patterns in type 1 diabetes (T1D) and breast cancer. This workflow consists of population-level analysis, followed by donor-level analysis, data transformation such as stratification or normalization, and a return to population-level analysis. In the T1D study, T-cell cytokine production was measured with a cytokine bead array. In the breast cancer study, intracellular cytokine staining measured T cell responses to stimulation with a variety of antigens. Summary statistics from each study were loaded into a relational database, together with associated experimental metadata and clinical parameters. Visual and statistical results were generated with custom Java software. In the T1D study, donor-level analysis led to the stratification of donors based on unstimulated cytokine expression. The resulting cohorts showed statistically significant differences in poststimulation production of IL-10, IL-1 beta, IL-8, and TNF beta. In the breast cancer study, the differing magnitude of cytokine responses required data normalization to support statistical comparisons. Once normalized, data showed a statistically significant decrease in the expression of IFN gamma on CD4+ and CD8+ T cells when stimulated with tumor-associated antigens (TAAs) when compared with an infectious disease antigen stimulus, and a statistically significant increase in expression of IL-2 on CD8+ T cells. In conclusion, the analytical workflow described herein yielded statistically supported and biologically relevant findings that were otherwise unapparent.

    View details for DOI 10.1002/cyto.a.20509

    View details for Web of Science ID 000254699000004

  • Precision and linearity targets for validation of an IFN gamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides BMC IMMUNOLOGY Maecker, H. T., Hassler, J., Payne, J. K., Summers, A., Comatas, K., Ghanayem, M., Morse, M. A., Clay, T. M., Lyerly, H. K., Bhatia, S., Ghanekar, S. A., Maino, V. C., delaRosa, C., Disis, M. L. 2008; 9


    Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNgamma-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays.Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen.These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

    View details for DOI 10.1186/1471-2172-9-9

    View details for Web of Science ID 000254604100001

    View details for PubMedID 18366814

  • Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI) CANCER IMMUNOLOGY IMMUNOTHERAPY Janetzki, S., Janetzki, S., Panageas, K. S., Ben-Porat, L., Boyer, J., Britten, C. M., Clay, T. M., Kalos, M., Maecker, H. T., Romero, P., Yuan, J., Kast, W. M., Hoos, A. 2008; 57 (3): 303-315


    The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.

    View details for DOI 10.1007/s00262-007-0380-6

    View details for Web of Science ID 000251794100003

    View details for PubMedID 17721781

  • Bacillus Calmette-Guerin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles JOURNAL OF IMMUNOLOGY Soares, A. P., Scriba, T. J., Joseph, S., Harbacheuski, R., Murray, R. A., Gelderbloem, S. J., Hawkridge, A., Hussey, G. D., Maecker, H., Kaplair, G., Hanekom, W. A. 2008; 180 (5): 3569-3577


    The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.

    View details for Web of Science ID 000256730000099

    View details for PubMedID 18292584

  • Poor predictive value of cytomegalovirus (CMV)-specific T cell assays for the development of CMV retinitis in patients with AIDS CLINICAL INFECTIOUS DISEASES Jacobson, M. A., Tan, Q. X., Girling, V., Poon, C., Van Natta, M., Jabs, D. A., Inokuma, M., Maecker, H. T., Bredt, B., Sinclair, E. 2008; 46 (3): 458-466


    We examined the potential clinical utility of using a cytomegalovirus (CMV)-specific T cell immunoassay to determine the risk of developing new-onset CMV retinitis (CMVR) in patients with acquired immunodeficiency syndrome (AIDS).CMV-specific T cell assays were performed by multiparameter flow cytometry using stored peripheral blood mononuclear cells that had been obtained in an observational study 2-6 months before new-onset CMVR was diagnosed in case patients (at a study visit during which a dilated ophthalmologic examination revealed no evidence of CMVR) and at the same study visit in control subjects (matched by absolute CD4(+) T cell count at entry) who did not subsequently develop retinitis during 1-6 years of study follow-up.There were no significant differences in CMV-specific CD4(+) or CD8(+) T cell interferon-gamma or interleukin-2 expression in peripheral blood mononuclear cells from case patients and control subjects. Although there were trends toward lower percentages and absolute numbers of CMV-specific, cytokine-expressing CD8(+) T cells with a "late memory" phenotype (CD27(-)CD28(-)) as well as with an "early memory" phenotype (CD27(+)CD28(+)CD45RA(+)) in case patients than in control subjects, these differences were not statistically significant.Many studies have reported that CMV-specific CD4(+) and CD8(+) T cell responses distinguish patients with active CMVR (i.e., who lack CMV-protective immunity) from those with inactive CMVR after immune restoration by antiretroviral treatment (i.e., who have CMV-protective immunity). However, the multiple CMV-specific immune responses we measured do not appear to have clinical utility for predicting the risk for patients with AIDS of developing new-onset CMVR with sufficient accuracy to be used in guiding therapeutic management.

    View details for DOI 10.1086/525853

    View details for Web of Science ID 000252221200018

    View details for PubMedID 18173357

  • Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature JOURNAL OF IMMUNOLOGY Inokuma, M., dela Rosa, C., Schmitt, C., Haaland, P., Siebert, J., Petry, D., Tang, M., Suni, M. A., Ghanekar, S. A., Gladding, D., Dunne, J. F., Maino, V. C., Disis, M. L., Maecker, H. T. 2007; 179 (4): 2627-2633


    The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8(+) T cells, and a lower proportion of IFN-gamma-producing CD4(+) and/or CD8(+) T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8(+) T cells in breast cancer patients was almost completely CD28(+)CD45RA(-) (memory phenotype). CMV-responsive CD8(+) T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27(-)CD28(-)CD45RA(+)) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.

    View details for Web of Science ID 000248959200069

    View details for PubMedID 17675526

  • Loss of Receptor on Tuberculin-Reactive T-Cells Marks Active Pulmonary Tuberculosis PLOS ONE Streitz, M., Tesfa, L., Yildirim, V., Yahyazadeh, A., Ulrichs, T., Lenkei, R., Quassem, A., Liebetrau, G., Nomura, L., Maecker, H., Volk, H., Kern, F. 2007; 2 (8)


    Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells.Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naïve/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between.Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

    View details for DOI 10.1371/journal.pone.0000735

    View details for Web of Science ID 000207455200012

    View details for PubMedID 17710135

  • Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors. Journal of immune based therapies and vaccines Ghanekar, S. A., Bhatia, S., Ruitenberg, J. J., dela Rosa, C., Disis, M. L., Maino, V. C., Maecker, H. T., Waters, C. A. 2007; 5: 7-?


    Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors.Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFalpha+IL-1beta+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells.Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro.Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.

    View details for PubMedID 17477875

  • Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon-G and interleukin-2 and have a CD8(+) cell early maturational phenotype JOURNAL OF INFECTIOUS DISEASES Sinclair, E., Tan, Q. X., Sharp, M., Girling, V., Poon, C., Van Natta, M., Jabs, D. A., Inokuma, M., Maecker, H. T., Bredt, B., Jacobson, M. A. 2006; 194 (11): 1537-1546


    To determine potential correlates of immune recovery from AIDS-related cytomegalovirus retinitis (CMVR), multiparameter flow cytometry was used to characterize CMV-specific T cells from subjects with CMVR. Individuals with active retinitis were compared with those who had been clinically immunorestored by antiretroviral therapy and had > or =2 years of ophthalmologic follow-up without anti-CMV therapy or retinitis reactivation or progression. In comparison with patients with active retinitis, immunorestored patients had higher circulating CD4(+) and CD8(+) T cells expressing interleukin-2 and interferon- gamma in response to combined CMV pp65 and IE1 peptide pool stimulation. CD4(+) T cell responses were predominantly to pp65, whereas CD8(+) T cell responses were predominantly to IE. Immunorestored patients, compared with patients with active retinitis, had increased levels of circulating CMV-specific CD8(+) T cells with "early" (CD27(+)CD28(+)CD45RA(+), CD27(+)CD28(+)CD45RA(-)) and "intermediate" (CD27(-)CD28(+)CD45RA(-)) phenotypes. Recovery from AIDS-related CMVR after the initiation of antiretroviral therapy may be mediated by CMV-specific CD4(+) and CD8(+) T cells capable of promoting antigen-specific CD8(+) T cell proliferation.

    View details for Web of Science ID 000241820800010

    View details for PubMedID 17083038

  • Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8(+) T cell response JOURNAL OF IMMUNOLOGY Murray, R. A., Mansoor, N., Harbacheuski, R., Soler, J., Davids, V., Soares, A., Hawkridge, A., Hussey, G. D., Maecker, H., Kaplan, G., Hanekom, W. A. 2006; 177 (8): 5647-5651


    Mounting evidence points to CD8+ T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Guérin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8+ T cell response. These cells degranulated or secreted IFN-gamma, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8+ T cell response was quantitatively smaller than the BCG-induced CD4+ T cell response. Incubation of whole blood with M. tuberculosis also caused CD8+ T cell IFN-gamma expression. We conclude that BCG induces a robust CD8+ T cell response, which may contribute to vaccination-induced protection against tuberculosis.

    View details for Web of Science ID 000241093100080

    View details for PubMedID 17015753

  • Flow cytometry controls, instrument setup, and the determination of positivity CYTOMETRY PART A Maecker, H. T., Trotter, J. 2006; 69A (9): 1037-1042


    A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option.

    View details for DOI 10.1002/cyto.a.20333

    View details for Web of Science ID 000240831500011

  • Immune responses in the draining lymph nodes against cancer: Implications for immunotherapy CANCER AND METASTASIS REVIEWS Shu, S., Cochran, A. J., Huang, R., Morton, D. L., Maecker, H. T. 2006; 25 (2): 233-242


    Regional lymph nodes are the first site for melanoma metastases. The sentinel node (SN), on the direct lymphatic drainage pathway, which usually harbors first metastases, demonstrates significant suppression in its ability to respond to antigenic stimulation. This down-regulation of SN immunity is likely the basis of its susceptibility to tumor metastases, suggesting a potential role of the immune system in the control of malignant tumors. Despite immune dysfunction in the SN, phase II trials of systemic post-operative immunotherapy with a polyvalent melanoma vaccine developed at the John Wayne Cancer Institute showed improved 5-year overall survival in patients with melanoma metastatic to regional nodes. However, most immunotherapy clinical trials have failed to demonstrate a significant clinical response, and analyses of immune responses to tumor-associated antigens that correlate clinical responses have not been established. Therefore, refinements in assay methodologies and improvements in vaccine designs are critical to the success of cancer immunotherapy. Antigen presentation by dendritic cells (DCs) is the most potent means to initiate a T cell immunity. Dendritic cell-based immunotherapies have been vigorously attempted in the past decade. To improve the immunogenicity of cancer vaccines, we recently generated heterokaryons of DCs and tumor cells by electrofusion. The fusion hybrids retained their full antigen-presenting capacity and all natural tumor antigens. In pre-clinical animal experiments, a single injection of the DC-tumor fusion hybrids was sufficient to mediate the regression of tumors established in the lung, skin and brain. Most interestingly, successful therapy required the delivery of fusion hybrids directly into lymphoid organs such as lymph nodes. A clinical trial is now being carried out to test the immunogenicity and therapeutic effects of fusion hybrids for the treatment of metastatic melanoma.

    View details for DOI 10.1007/s10555-006-8503-7

    View details for Web of Science ID 000238268800006

    View details for PubMedID 16770535

  • Maximizing the retention of antigen specific lymphocyte function after cryopreservation JOURNAL OF IMMUNOLOGICAL METHODS Disis, M. L., dela Rosa, C., Goodell, V., Kuan, L. Y., Chang, J. C., Kuus-Reichel, K., Clay, T. M., Lyerly, H. K., Bhatia, S., Ghanekar, S. A., Maino, V. C., Maecker, H. T. 2006; 308 (1-2): 13-18


    The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.

    View details for DOI 10.1016/j.jim.2005.09.011

    View details for Web of Science ID 000235285700002

    View details for PubMedID 16337957

  • IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells. AIDS research and therapy Nomura, L. E., Emu, B., Hoh, R., Haaland, P., Deeks, S. G., Martin, J. N., McCune, J. M., Nixon, D. F., Maecker, H. T. 2006; 3: 18-?


    Diminished IL-2 production and lack of effector differentiation have been reported for HIV-specific T cells. In this study, we examined the prevalence of these phenomena using 8-color cytokine flow cytometry, and tested the hypothesis that these two findings were causally related. We analyzed cytokine profiles and memory/effector phenotypes of HIV-specific and CMV-specific T cells using short-term in vitro stimulation with HIV or CMV peptide pools. Nineteen HIV-positive subjects with progressive disease and twenty healthy, HIV-negative subjects were examined.Among HIV-infected subjects, there were significantly fewer CD8+ IL-2+ T cells responding to HIV compared to CMV, with no significant difference in CD4+ IL-2+ T cells. The majority of CMV-specific T cells in both HIV-negative and HIV-positive subjects appeared to be terminally differentiated effector cells (CD8+ CD27- CD28- CD45RA+ or CD8+ CD27- CD28- CD45RA-). In HIV-positive subjects, the most common phenotype of HIV-specific T cells was intermediate in differentiation (CD8+ CD27+ CD28- CD45RA-). These differences were statistically significant, both as absolute cell frequencies and as percentages. There was a significant correlation between the absolute number of HIV-specific CD8+ IL-2+ T cells and HIV-specific CD8+ CD27- CD28- CD45RA+ terminal effector cells.IL-2 production from antigen-specific CD8+ T cells correlates with effector cell differentiation of those cells.

    View details for PubMedID 16859558

  • Ex vivo analysis of T-cell function CURRENT OPINION IN IMMUNOLOGY Suni, M. A., Maino, V. C., Maecker, H. T. 2005; 17 (4): 434-440


    Our ability to analyze T-cell function in vitro has progressed in recent years to include analysis of early signaling events, such as specific protein phosphorylation, intermediate functions, such as degranulation and cytokine production, and later functions, such as proliferation. Many assays are now available to monitor these events, and comparative studies of some of these assays have been published. Major recent developments in this area include the ability to measure T-cell degranulation via cell surface exposure of CD107 and the use of polychromatic flow cytometry to examine multiple phenotypes and functions of responding T cells.

    View details for DOI 10.1016/j.coi.2005.05.002

    View details for Web of Science ID 000230673900016

    View details for PubMedID 15950444

  • Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT BMC IMMUNOLOGY Maecker, H. T., Moon, J., Bhatia, S., Ghanekar, S. A., Maino, V. C., Payne, J. K., Kuus-Reichel, K., Chang, J. C., Summers, A., Clay, T. M., Morse, M. A., Lyerly, H. K., DeLaRosa, C., Ankerst, D. P., Disis, M. L. 2005; 6


    Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors.Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p

    View details for DOI 10.1186/1471-2172-6-17

    View details for Web of Science ID 000235714000001

    View details for PubMedID 16026627

  • Standardization of cytokine flow cytometry assays BMC IMMUNOLOGY Maecker, H. T., Rinfret, A., D'Souza, P., Darden, J., Roig, E., Landry, C., Hayes, P., Birungi, J., Anzala, O., Garcia, M., Harari, A., Frank, I., Baydo, R., Baker, M., Holbrook, J., Ottinger, J., Lamoreaux, L., Epling, C. L., Sinclair, E., Suni, M. A., Punt, K., Calarota, S., El-Bahi, S., Alter, G., Maila, H., Kuta, E., Cox, J., Gray, C., Altfeld, M., Nougarede, N., Boyer, J., Tussey, L., Tobery, T., Bredt, B., Roederer, M., Koup, R., Maino, V. C., Weinhold, K., Pantaleo, G., Gilmour, J., Horton, H., Sekaly, R. P. 2005; 6


    Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

    View details for DOI 10.1186/1471-2172-6-13

    View details for Web of Science ID 000235713700001

    View details for PubMedID 15978127

  • Immune dysfunction and micrometastases in women with breast cancer BREAST CANCER RESEARCH AND TREATMENT Campbell, M. J., Scott, J., Maecker, H. T., Park, J. W., Esserman, L. J. 2005; 91 (2): 163-171


    Cytokines produced by T lymphocytes are critical to the efficacy of a given immune response and dysregulation of immune responses may play a role in cancer progression. We assessed the intracellular cytokine profiles of T cells in the peripheral blood of women with breast cancer and explored the relationship of these responses with the presence of cancer in lymph nodes and bone marrow. Peripheral blood lymphocytes from 84 patients and 26 healthy volunteers were analyzed by 4-color flow cytometry for surface markers and for intracellular cytokines. Bone marrow samples from some of these patients were also collected and analyzed for the presence of epithelial cells (micrometastases) by flow cytometry. The percentages of both CD4(+) and CD8(+) cells producing type1 (IL-2, IFN-gamma or TNF-alpha) and type 2 (IL-4) were significantly lower in patients with breast cancer compared to healthy controls. These results indicate a general immune dysfunction in these patients as opposed to a shift in the balance of type1 and type2 cells. These dysregulated T cell responses did not correlate with age, stage of disease, or nodal status. However, we did observe a correlation between number of micrometastases in the bone marrow and T cell responsiveness.

    View details for DOI 10.1007/s10549-004-7048-0

    View details for Web of Science ID 000228867600008

    View details for PubMedID 15868444

  • Dysfunction of simian immunodeficiency virus/simian human immunodeficiency virus-induced IL-2 expression by central memory CD4+ T lymphocytes JOURNAL OF IMMUNOLOGY Sun, Y., Schmitz, J. E., Acierno, P. M., Santra, S., Subbramanian, R. A., Barouch, D. H., Gorgone, D. A., Lifton, M. A., Beaudry, K. R., Manson, K., Philippon, V., Xu, L., Maecker, H. T., MASCOLA, J. R., Panicali, D., Nabel, G. J., Letvin, N. L. 2005; 174 (8): 4753-4760


    Production of IL-2 and IFN-gamma by CD4+ T lymphocytes is important for the maintenance of a functional immune system in infected individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4+ T lymphocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates, and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following pathogenic SHIV challenge. We observed that preserved central memory CD4+ T lymphocyte production of SIV/SHIV-induced IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4+ T cells to express this important immunomodulatory cytokine.

    View details for Web of Science ID 000228234600040

    View details for PubMedID 15814700

  • Selecting fluorochrome conjugates for maximum sensitivity CYTOMETRY PART A Maecker, H. T., Frey, T., Nomura, L. E., Trotter, J. 2004; 62A (2): 169-173
  • Selective developmental defects of cord blood antigen-presenting cell subsets HUMAN IMMUNOLOGY Drohan, L., Harding, J. J., Holm, B., Cordoba-Tongson, E., Dekker, C. L., Holmes, T., Maecker, H., Mellins, E. D. 2004; 65 (11): 1356-1369


    Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased infection susceptibility in newborns. We used multiparameter flow cytometry to characterize APC subsets in adult peripheral blood (APB) and cord blood (CB). APB had a higher proportion of CD11c+ dendritic cells (DC), whereas CB mainly contained CD123+ DC. APB was enriched in CD16+CD11c+ DC subset, whereas CD34+CD11c-CD123lo cells were prominent in CB. Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was dampened in myeloid DC and monocytes from CB, whereas IL-1alpha production was not different. The reduction in TNF-alpha response did not appear to result from reduced surface detection of LPS, because CD14, toll-like receptor (TLR)-4 and TLR-2 levels were not reduced in CB APC compared with APB cells. Also, there was no correlation between TLR-2 or TLR-4 levels and TNF-alpha production in myeloid DC and monocytes. CB monocytes had lower surface HLA-DR immediately ex vivo. Both APB and CB monocytes upregulated HLA-DR after incubation, but an additional LPS-induced increase in HLA-DR was suggested only in APB monocytes. APB monocytes also showed a greater LPS-induced increase in CD40 expression. Together, our data show significant, selective differences in circulating APC between neonates and adults.

    View details for DOI 10.1016/j.humimm.2004.09.011

    View details for Web of Science ID 000225728800010

    View details for PubMedID 15556686

  • Analyzing T-cell responses to cytomegalovirus by cytokine flow cytometry HUMAN IMMUNOLOGY Maecker, H. T., Maino, V. C. 2004; 65 (5): 493-499


    T-cell responses to human cytomegalovirus (CMV) are readily detected in chronically infected adults, and are thought to be important for protection from CMV-related pathology. Antigen-specific cytokine flow cytometry (CFC) has been used to establish the range of CMV-specific CD4 and CD8 T-cell frequencies in healthy CMV-seropositive (and seronegative) adults, as well as the dynamics of these cells over time. There are also emerging data regarding the primary CD4 and CD8 T-cell response to CMV in children and adults. Finally, CFC has been used to analyze CMV responses in chronic human immunodeficiency virus infection, as well as during immune reconstitution after bone marrow or stem cell transplantation. These data will be reviewed in terms of what they suggest about the threshold of protective T-cell immunity to CMV, and other factors in addition to T-cell frequencies that could be important in protecting from CMV-associated disease.

    View details for Web of Science ID 000222232400015

    View details for PubMedID 15172449

  • Results of a cytomegalovirus (CMV)-specific CD8(+)/interferon-gamma(+) cytokine flow cytometry assay correlate with clinical evidence of protective immunity in patients with AIDS with CMV retinitis JOURNAL OF INFECTIOUS DISEASES Jacobson, M. A., Maecker, H. T., Orr, P. L., D'Amico, R., Van Natta, M., Li, X. D., Pollard, R. B., Bredt, B. M. 2004; 189 (8): 1362-1373


    To evaluate the potential clinical utility of a cytomegalovirus (CMV)-specific CD8+/interferon (IFN)- gamma+ cytokine flow cytometry (CFC) assay for patients with CMV retinitis (CMVR), stored peripheral blood mononuclear cell specimens were obtained from patients with active CMVR (i.e., having clinical evidence of absent CMV-protective immunity), as well as from highly active antiretroviral therapy-treated patients with CMVR who were able to discontinue anti-CMV therapy without subsequent progression of retinitis (i.e., having clinical evidence of restored CMV-protective immunity). Positive CD8+/IFN- gamma+ T lymphocyte responses to CMV phosphoprotein 65 or immediate early peptide-pool stimulation were present in specimens from only 3 of 10 patients with active CMVR but were present in at least 1 specimen from all 20 patients with immunorestored CMVR, with a mean of 2.4 specimens/patient tested, spanning up to 6 months of observation (P = .0001). Among the patients with immunorestored CMVR, positive responses were present in all longitudinal specimens from 15 of the 20 patients. These data suggest that further testing of the CMV-specific CD8+/IFN- gamma+ CFC assay, for clinical utility in predicting incident and progressive CMVR disease, is warranted.

    View details for Web of Science ID 000220735400005

    View details for PubMedID 15073672

  • Cytokine flow cytometry: a multiparametric approach for assessing cellular immune responses to viral antigens CLINICAL IMMUNOLOGY Maino, V. C., Maecker, H. T. 2004; 110 (3): 222-231


    Considerable attention has been focused on CD8 and CD4 T cell responses as a major element of the cellular immune response to viral infections including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, increasing evidence based on the recent introduction of more quantitative assays for measuring antigen-specific T cells has suggested that the role of these cells in the development of a protective immune response to a particular viral pathogen may be determined by a complex interplay of multiple virologic and cellular factors. Thus, measurements of only the frequencies of the T cell subsets participating in the response to viral pathogens may be an incomplete reflection of efficacy. In this review, we suggest that some measurable factors may influence the role of T cell immunity in conferring protection, including functional avidity, epitope breadth and specificity, proliferative capacity, cytokine repertoire, degree of anergy, and differentiation phenotype, as well as magnitude, of viral-specific CD4 and CD8 T cells. We suggest that automated cytokine flow cytometry (CFC) is an efficient approach to the measurement of the complex interplay of multiple immune parameters involved in immune protection. These ideas are discussed in the context of new developments in sample preparation and analysis automation.

    View details for DOI 10.1016/j.clim.2003.11.018

    View details for Web of Science ID 000220831800004

    View details for PubMedID 15047200

  • Persistent and selective deficiency of CD4(+) T cell immunity to cytomegalovirus in immunocompetent young children JOURNAL OF IMMUNOLOGY Tu, W. W., Chen, S., Sharp, M., Dekker, C., Manganello, A. M., Tongson, E. C., Maecker, H. T., Holmes, T. H., Wang, Z. T., Kemble, G., Adler, S., Arvin, A., Lewis, D. B. 2004; 172 (5): 3260-3267


    Healthy young children who acquire CMV have prolonged viral shedding into the urine and saliva, but whether this is attributable to limitations in viral-specific immune responses has not been explored. In this study, we found that otherwise immunocompetent young children after recent primary CMV infection accumulated markedly fewer CMV-specific CD4(+) T cells that produced IFN-gamma than did adults. These differences in CD4(+) T cell function persisted for more than 1 year after viral acquisition, and did not apply to CMV-specific IFN-gamma production by CD8(+) T cells. The IFN-gamma-producing CD4(+) T cells of children or adults that were reactive with CMV Ags were mainly the CCR7(low) cell subset of memory (CD45R0(high)CD45RA(low)) cells. The decreased IFN-gamma response to CMV in children was selective, because their CCR7(low) memory CD4(+) T cells and those of adults produced similar levels of this cytokine after stimulation with staphylococcal enterotoxin B superantigen. CD4(+) T cells from children also had reduced CMV-specific IL-2 and CD154 (CD40 ligand) expression, suggesting an early blockade in the differentiation of viral-specific CD4(+) T cells. Following CMV acquisition, children, but not adults, persistently shed virus in urine, and this was observable for at least 29 mo postinfection. Thus, CD4(+) T cell-mediated immunity to CMV in humans is generated in an age-dependent manner, and may have a substantial role in controlling renal viral replication and urinary shedding.

    View details for Web of Science ID 000189186000069

    View details for PubMedID 14978134

  • Strong cell-mediated immune responses are associated with the maintenance of low-level viremia in antiretroviral-treated individuals with drug-resistant human immunodeficiency virus type 1 JOURNAL OF INFECTIOUS DISEASES Deeks, S. G., Martin, J. N., Sinclair, E., Harris, J., Neilands, T. B., Maecker, H. T., Hagos, E., Wrin, T., Petropoulos, C. J., Bredt, B., McCune, J. M. 2004; 189 (2): 312-321


    Antiretroviral (ARV)-treated patients often maintain low to moderate levels of viremia, despite the emergence of drug-resistant human immunodeficiency virus (HIV). We studied host and viral factors that may contribute to the control of viral replication in a cohort of 189 adults. Among ARV-treated patients with detectable viremia, there was a bell-shaped relationship between Gag-specific CD4+ T cell responses and viremia, with the highest cellular immune responses observed in patients with plasma HIV RNA levels of 1000-10,000 copies/mL. In contrast, there was a negative association between Gag-specific CD4+ T cell responses and viremia among ARV-untreated individuals with wild-type HIV. Strong cellular immune responses among individuals with drug-resistant HIV predicted subsequent lack of virological progression. Finally, there was a positive correlation between replicative capacity and viremia. Collectively, these data suggest that the selection of drug-resistance mutations may reduce the pathogenic potential of HIV, which leads to a balanced state of enhanced cellular immunity and low-level viremia.

    View details for Web of Science ID 000188097900019

    View details for PubMedID 14722897

  • gp100(209-2M) peptide immunization of human lymphocyte antigen-A2(+) stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8(+) T cells CLINICAL CANCER RESEARCH Walker, E. B., Haley, D., Miller, W., Floyd, K., Wisner, K. P., Sanjuan, N., Maecker, H., Romero, P., Hu, H. M., Alvord, W. G., Smith, J. W., Fox, B. A., Urba, W. J. 2004; 10 (2): 668-680


    Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

    View details for Web of Science ID 000188801100033

    View details for PubMedID 14760090

  • Cytokine flow cytometry. Methods in molecular biology (Clifton, N.J.) Maecker, H. T. 2004; 263: 95-108


    Cytokine flow cytometry (CFC) is a general term that applies to flow cytometric analysis of cells using anticytokine antibodies as markers of activation. The most common version of this technique is the intracellular staining of cytokines in cells that have been fixed and permeabilized after short-term in vitro activation. When used with specific antigens, this technique allows for the quantitation of rare populations of antigen-specific T cells. In this chapter, specific methodology for such intracellular staining is elaborated, with emphasis on the effects of variables such as sample type, antigens, activation conditions, sample processing, and data acquisition and analysis.

    View details for PubMedID 14976362

  • Multicolor flow cytometric analysis in SIV-infected rhesus macaque CYTOMETRY, 4TH EDITION: NEW DEVELOPMENTS Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. 2004; 75: 535-557

    View details for Web of Science ID 000227570400022

    View details for PubMedID 15603441

  • TNF-alpha detection using a flow cytometric assay to detennine cellular responses to anthrax vaccine JOURNAL OF IMMUNOLOGICAL METHODS Shinn, A. H., Bravo, N. C., Maecker, H. T., Smith, J. W. 2003; 282 (1-2): 169-174


    This study describes a four-color flow cytometric assay that detects CD4+ T cell responses to the anthrax vaccine. Whole blood from seven volunteers who previously obtained the anthrax vaccine was inoculated in vitro with varying concentrations of the anthrax antigen. TNF-alpha and IFN-gamma production from memory CD4+ T cells were measured and compared to a control group who never received the anthrax vaccine. The optimal antigen concentration for TNF-alpha was determined to be around 7.5 microg/ml and IFN-gamma production was not detected. This assay will be used in future larger prospective studies to further evaluate the cellular immune responses induced by the anthrax vaccine.

    View details for DOI 10.1016/j.jim.2003.07.012

    View details for Web of Science ID 000186726400015

    View details for PubMedID 14604550

  • Performance of plate-based cytokine flow cytometry with automated data analysis. BMC immunology Suni, M. A., Dunn, H. S., Orr, P. L., de laat, R., Sinclair, E., Ghanekar, S. A., Bredt, B. M., Dunne, J. F., Maino, V. C., Maecker, H. T. 2003; 4: 9-?


    Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates.We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis.When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.

    View details for PubMedID 12952557

  • Optimal preparation of rhesus macaque blood for cytokine flow cytometric analysis CYTOMETRY PART A Nomura, L. E., deHaro, E. D., Martin, L. N., Maecker, H. T. 2003; 53A (1): 28-38


    The rhesus macaque is a common substitute for human subjects in many disease models, including simian immunodeficiency virus, the non-human primate equivalent of the human immunodeficiency virus. Monoclonal antibodies and fluorochromes optimized for use in macaques were included in samples examined for immune responses with the use of intracellular cytokine flow cytometry (CFC).Sample preparation was optimized based on the following comparisons: activation of peripheral blood mononuclear cells (PBMCs) versus whole blood; separation of PBMCs using BD Vacutainer cell preparation tubes versus Ficoll; and activation of samples on the day they were collected versus holding samples overnight.When activated with the simian immunodeficiency virus type mac239 and Gag peptide mix or with the superantigen Staphylococcal enterotoxin B, separated PBMCs produced greater CD4 and CD8 fluorescence intensities and a larger percentage of CD69+ cytokine-positive cells than did whole blood samples. PBMCs separated by cell preparation tubes produced absolute T-lymphocyte counts equivalent to that with Ficoll separation, and CFC results with both methods were similar. When subjected to overnight shipping conditions, whole blood and PBMCs sometimes showed a reduction in mean fluorescence intensity and percentage of CD69+ cytokine-positive T lymphocytes.Due to this reduction in responses, it is preferable to activate samples on the day of blood collection. Samples can be surface stained and frozen in BD FACS Lysing Solution, to be thawed at a later date; this preserves their ability to display a cytokine response. Thus optimal CFC results are achieved by separating macaque PBMCs from whole blood, activating samples on day of collection, and, if necessary, freezing samples after surface staining for future analysis.

    View details for DOI 10.1002/cyto.a.10038

    View details for Web of Science ID 000182718000004

  • Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8(+) T-cell responses JOURNAL OF CLINICAL ONCOLOGY Smith, J. W., Walker, E. B., Fox, B. A., Haley, D., Wisner, K. P., Doran, T., Fisher, B., Justice, L., Wood, W., Vetto, J., Maecker, H., Dols, A., Meijer, S., Hu, H. M., Romero, P., Alvord, W. G., Urba, W. J. 2003; 21 (8): 1562-1573


    To measure the CD8+ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule.Thirty HLA-A2-positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209-2M, and a control peptide, HPV16 E7, were mixed in incomplete Freund's adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry.Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8+ T cells specific for the g209-2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P <.0001). Eight patients (28%) exhibited peptide-specific CD8+ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b-treated patients also had significant increases in tetramer-binding cells (P <.0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P =.59).Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8+ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.

    View details for DOI 10.1200/JCO.2003.09.020

    View details for Web of Science ID 000182300200022

    View details for PubMedID 12697882

  • Analysis of the frequencies and of the memory T cell phenotypes of human CD8(+) T cells specific for influenza A viruses JOURNAL OF INFECTIOUS DISEASES He, X. S., Mahmood, K., Maecker, H. T., Holmes, T. H., Kemble, G. W., Arvin, A. M., Greenberg, H. B. 2003; 187 (7): 1075-1084


    We characterized the human CD8+ T cell response against influenza A viruses by a flow cytometry-based assay. Peripheral blood mononuclear cells (PBMCs) were incubated with inactivated influenza virus preparation, for 17 h, and were stained for intracellular interferon-gamma. Major histocompatibility complex class I-restricted memory CD8+ T cells specific for influenza antigens were detected in PBMCs from all 19 adult donors, at an average frequency of 0.39%. On average, 83% of influenza virus-specific CD8+ T cells expressed the differentiation-associated marker CD27, a percentage that is significantly higher than that of CD8+ T cells specific for pp65 of human cytomegalovirus (53%). These observations indicate that class I-restricted immunity against influenza A viruses is characterized by the persistence, after clearance of infection, of circulating antigen-specific CD8+ T cells. The different patterns of CD27 expression in influenza virus- and cytomegalovirus-specific CD8+ T cells suggest that influenza virus-specific memory and effector CD8+ T cells can be differentiated by phenotypic analysis.

    View details for Web of Science ID 000181713000007

    View details for PubMedID 12660922

  • T cell immunity to HIV: Defining parameters of protection CURRENT HIV RESEARCH Maecker, H. T., Maino, V. C. 2003; 1 (2): 249-259


    In recent years, CD4 and CD8 T cell responses to HIV and SIV infection have been increasingly measured with the use of single-cell assays such as ELISPOT, MHC-peptide oligomers, and cytokine flow cytometry. The results of these assays have been compared to those obtained with traditional bulk assays such as lymphoproliferation (by 3H-thymidine incorporation) and cytotoxicity (by 51Cr release). Such comparisons have led to some general understanding of the T cell responses that characterize progressive disease, long-term non-progressors, and individuals with viral suppression achieved by anti-retroviral therapy. In addition, prophylactic and therapeutic vaccine trials have also begun to use these assays of T cell immunity to gauge the immunogenicity of the vaccines. Whether such analyses will allow us to pick the best vaccine constructs, and whether they will provide us with an improved understanding of what constitutes protective cellular immunity to HIV, are major questions for the field. These questions will be examined in this review from the standpoint of current data and comparisons to other viral diseases. It is hypothesized that sophisticated multiparametric assays will be required to sort out the factors relevant for protective immunity in this complex disease. These parameters may include functional avidity, epitope breadth and specificity, proliferative capacity, cytokine repertoire, degree of anergy, and differentiation phenotype, as well as magnitude, of HIV-specific CD4 and CD8 T cells.

    View details for Web of Science ID 000187935400009

    View details for PubMedID 15043207

  • Human CD81 directly enhances Th1 and Th2 cell activation, but preferentially induces proliferation of Th2 cells upon long-term stimulation. BMC immunology Maecker, H. T. 2003; 4: 1-?


    CD81, a cell-surface protein of the tetraspanin superfamily, has been shown to costimulate T cell activation in murine T cells, and is involved in development of Th2 immune responses in mice.Here it is shown that stimulation of CD81 on human T cells can enhance T cell activation by antigen or superantigen, causing an increase in the early activation marker CD69, and increasing the number of cytokine-producing and proliferating T cells. Interestingly, CD81 costimulates cytokine production by T cells producing both Th1 and Th2 cytokines. Although human CD81 is highly expressed on non-T as well as T cells, CD81 costimulation appears to act directly on T cells. Pre-incubation of purified T cells with anti-CD81 antibody is sufficient to increase T cell activation, while pre-incubation of non-T cells is not. However, long-term polyclonal stimulation of T cells by anti-CD3 antibody, in the presence of CD81 costimulation, biases T cells towards the production of IL-4 and not IFNgamma. This is accomplished by a preferential proliferation of IL-4-producing cells.Thus, signalling through CD81 on T cells costimulates both Th1 and Th2 cells, but increases the number of Th2 cells during long-term activation.

    View details for PubMedID 12597781

  • Cytokine flow cytometry: multiparametric approach to immune function analysis CYTOTHERAPY Ghanekar, S. A., Maecker, H. T. 2003; 5 (1): 1-6


    More precise quantitation of cellular immune responses has become possible with the advent of single-cell assays of immune function, such as cytokine flow cytometry, enzyme-linked immunospot (ELISPOT), and MHC-peptide multimers. Cytokine flow cytometry is an attractive technique because it allows the detection of responses to whole antigens without regard to MHC restriction, while also collecting additional information on responding cells via multiparameter flow cytometry. In this review, we compare cytokine flow cytometry with other assays of immune function, summarize some of that data that have been collected in various disease states using cytokine flow cytometry, and describe some methodological improvements designed to increase the robustness, throughput, and information content of this technique. We hypothesize that a new generation of automated cytokine flow cytometry assays will allow elucidation of the correlates of protection for diseases involving cellular immunity, through application of these assays in more and large clinical trials.

    View details for DOI 10.1080/14653240310000029

    View details for Web of Science ID 000181768100001

    View details for PubMedID 12745590

  • Increased brain size and glial cell number in CD81-null mice JOURNAL OF COMPARATIVE NEUROLOGY Geisert, E. E., Williams, R. W., Geisert, G. R., Fan, L., Asbury, A. M., Maecker, H. T., Deng, J., Levy, S. 2002; 453 (1): 22-32


    A key issue in the development of the central nervous system (CNS) is understanding the molecular mechanisms regulating cell number. The present study examines the role of CD81 (previously known as TAPA, the target of the antiproliferative antibody) in the control of brain size and glial cell number. CD81 is a member of the tetraspanin family of proteins. This group of small membrane proteins is associated with the regulation of cell migration and mitotic activity. Glial cells express CD81, and antibodies directed against this protein suppress the mitotic activity of cultured cells. In this study, we examine the effects of the CD81 -/- mutation on the CNS of mature mice. These mice have extremely large brains, as much as 30% larger than the brains of wild-type (+/+) littermates. The increase in brain weight is accompanied by an increase in the number astrocytes and microglia, whereas the number of neurons and oligodendrocytes in the CD81 -/- animals appears to be normal. When the CD81 -/- mutation is placed on different genetic backgrounds, there is a remarkable range in the penetrance of the null allele phenotype, demonstrating that the mutation can be affected by modifier loci. This work provides support for the role of CD81 in the regulation of astrocyte and microglial number, perhaps by regulating cell proliferation by a contact inhibition-dependent mechanism.

    View details for DOI 10.1002/cne.10364

    View details for Web of Science ID 000178399900003

    View details for PubMedID 12357429

  • Epigenetic changes in tumor Fas levels determine immune escape and response to therapy CANCER CELL Maecker, H. L., Yun, Z., Maecker, H. T., Giaccia, A. J. 2002; 2 (2): 139-148


    Epigenetic regulation of gene expression significantly influences cell growth and differentiation. Here we show that epigenetic silencing of Fas determines tumor growth in vivo and apoptotic sensitivity in vitro. In established tumors with epigenetically repressed Fas, restoration of Fas activity either by transfection of fas or treatment with Trichostatin A (TSA), an inhibitor of histone deacetylase, suppresses tumor growth and restores chemosensitivity. The TSA-dependent chemosensitivity and tumor growth control require both tumor Fas and the host NK (natural killer) cell functions. This work demonstrates the importance of epigenetic modification of Fas in tumor progression and immune evasion, and emphasizes the essential interplay between Fas and innate immunity in the control of chemoresistant tumors.

    View details for Web of Science ID 000178352600009

    View details for PubMedID 12204534

  • Dynamics of CD4 and CD8 T cell responses to cytomegalovirus in healthy human donors JOURNAL OF INFECTIOUS DISEASES Dunn, H. S., Haney, D. J., Ghanekar, S. A., Stepick-Biek, P., Lewis, D. B., Maecker, H. T. 2002; 186 (1): 15-22


    To study the dynamics of cytomegalovirus (CMV) immunity in healthy immunocompetent hosts, interferon-gamma-producing CD4 and CD8 T cell responses in the presence or absence of CMV antigens were measured from 15 CMV-seropositive donors and 13 CMV-seronegative donors. Cytokine responses in the absence of antigen were significantly higher in CMV-seropositive donors. Also, a disproportionate number of CD69(+) cells isolated ex vivo from CMV-seropositive donors were specific for CMV, suggesting recent reactivation in vivo. To examine changes in cellular responses over time, 10 seropositive donors were tested over a 6-month period. About half of the donors showed significant variability over time, but staphylococcal enterotoxin B responses remained relatively constant. These findings suggest that CMV can present a considerable and recurrent burden to the human immune system. By understanding the normal dynamics of CMV responses over time, it may be possible to better identify aberrant responses to CMV in immunocompromised hosts.

    View details for Web of Science ID 000176307800003

    View details for PubMedID 12089657

  • Use of overlapping peptide mixtures as antigens for cytokine flow cytometry JOURNAL OF IMMUNOLOGICAL METHODS Maecker, H. T., Dunn, H. S., Suni, M. A., Khatamzas, E., Pitcher, C. J., Bunde, T., Persaud, N., Trigona, W., Fu, T. M., Sinclair, E., Bredt, B. M., McCune, J. M., Maino, V. C., Kern, F., Picker, L. J. 2001; 255 (1-2): 27-40


    Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

    View details for Web of Science ID 000170260300004

    View details for PubMedID 11470284

  • CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens EUROPEAN JOURNAL OF IMMUNOLOGY Suni, M. A., Ghanekar, S. A., Houck, D. W., Maecker, H. T., Wormsley, S. B., Picker, L. J., Moss, R. B., Maino, V. C. 2001; 31 (8): 2512-2520


    CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.

    View details for Web of Science ID 000170580500031

    View details for PubMedID 11500836

  • Factors affecting the efficiency of CD8(+) T cell cross-priming with exogenous antigens JOURNAL OF IMMUNOLOGY Maecker, H. T., Ghanekar, S. A., Suni, M. A., He, X. S., Picker, L. J., Maino, V. C. 2001; 166 (12): 7268-7275


    Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

    View details for Web of Science ID 000170949000034

    View details for PubMedID 11390476

  • Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY Ghanekar, S. A., Nomura, L. E., Suni, M. A., Picker, L. J., Maecker, H. T., Maino, V. C. 2001; 8 (3): 628-631


    Antigen-specific CD8(+) T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-gamma) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8(+) T-cell IFN-gamma expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a (51)Cr release assay and frequencies of peptide-activated CD8(+) T cells expressing IFN-gamma at 6 h (r(2) = 0.72) or 7 days (r(2) = 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-gamma expression can be a functional surrogate for identification of CTL precursor cells.

    View details for Web of Science ID 000168664400029

    View details for PubMedID 11329470

  • Vaccination with allergen-IL-18 fusion DNA protects against, and reverses established, airway hyperreactivity in a murine asthma model JOURNAL OF IMMUNOLOGY Maecker, H. T., Hansen, G., Walter, D. M., DeKruyff, R. H., Levy, S., Umetsu, D. T. 2001; 166 (2): 959-965


    Vaccination with naked DNA encoding a specific allergen has been shown previously to prevent, but not reverse, the development of allergen-induced airway hyperresponsiveness (AHR). To enhance the effectiveness of DNA vaccine therapies and make possible the treatment of established AHR, we developed a DNA vaccination plasmid containing OVA cDNA fused to IL-18 cDNA. Vaccination of naive mice either with this fusion DNA construct or with an OVA cDNA-containing plasmid protected the mice from the subsequent induction of AHR. Protection from AHR correlated with increased IFN-gamma production and reduced OVA-specific IgE production. The protection appeared to be mediated by IFN-gamma and CD8(+) cells because treatment of mice with neutralizing anti-IFN-gamma mAb or with depleting anti-CD8 mAb abolished the protective effect. Moreover, vaccination of mice with preexisting AHR with the OVA-IL-18 fusion DNA, but not with the OVA cDNA plasmid, reversed established AHR, reduced allergen-specific IL-4, and increased allergen-specific IFN-gamma production. Thus, combining IL-18 cDNA with OVA cDNA resulted in a vaccine construct that protected against the development of AHR, and that was unique among cDNA constructs in its capacity to reverse established AHR.

    View details for Web of Science ID 000166259600033

    View details for PubMedID 11145673

  • Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood VIRAL IMMUNOLOGY He, X. S., Rehermann, B., Boisvert, J., Mumm, J., Maecker, H. T., Roederer, M., Wright, T. L., Maino, V. C., DAVIS, M. M., Greenberg, H. B. 2001; 14 (1): 59-69


    The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.

    View details for Web of Science ID 000167575500005

    View details for PubMedID 11270597

  • Immunofluorescence analysis of T-cell responses in health and disease JOURNAL OF CLINICAL IMMUNOLOGY Maecker, H. T., Maino, V. C., Picker, L. J. 2000; 20 (6): 391-399


    The use of flow cytometry to study the functional responses of T cells by immunofluorescent staining for intracellular cytokines and other markers is a growing field of clinical interest. In this article, we describe methods for the rapid evaluation of T-cell responses to mitogens and specific antigens and explore how these assays might be valuable in various clinical settings.

    View details for Web of Science ID 000166556900001

    View details for PubMedID 11202228

  • Optimization of whole blood antigen-specific cytokine assays for CD4(+) cells CYTOMETRY Nomura, L. E., Walker, J. M., Maecker, H. T. 2000; 40 (1): 60-68


    The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4(+) T cells.We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors.CMV is most effective as a stimulating antigen when used at a dose of 5 microg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 microg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a "timed cooling" device, whereby the samples are automatically cooled and held at 4 degrees C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining.These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra-assay variability is less than 10%; interassay variability is less than 25%).

    View details for Web of Science ID 000086767900008

    View details for PubMedID 10754518

  • Differential expression of murine CD81 highlighted by new anti-mouse CD81 monoclonal antibodies HYBRIDOMA Maecker, H. T., Todd, S. C., Kim, E. C., Levy, S. 2000; 19 (1): 15-22


    We describe the use of a soluble CD81-Fc fusion protein to screen for novel monoclonal antibody (MAb) reactive with the extracellular loops of murine CD81 (TAPA-1). Two such MAbs, Eat1 and Eat2 (for Extracellular Anti-TAPA1), were used to assess the expression and function of CD81 on murine lymphocytes. Although CD81 is expressed uniformly on all human lymphocytes, murine CD81 was found to be expressed at much higher levels on resting B cells than on resting T cells. This was particularly evident when staining with the new MAbs, Eat1 and Eat2. The molecule is also functionally active on B cells, as Eat1 and Eat2 induce homotypic adhesion of B lymphocytes. Stimulated B cells undergo early apoptotic events in the presence of Eat2, as shown by binding of Annexin V-fluorescein isothiocyanate (FITC). Polyclonal activation of murine T cells also induces higher level CD81 expression, and many immortalized murine T-cell lines express high levels of the protein. In contrast to human CD81, which is expressed equally on all thymocytes, murine CD81 is induced during thymic development, being expressed at high levels on CD4+CD8+ thymocytes, in contrast to other subsets of thymocytes. Finally, murine dendritic cells, splenic macrophages, and non-killer (NK) cells all express high levels of CD81. We conclude that CD81 is differentially expressed in the murine immune system, and is involved in regulating the adhesion and activation of murine B cells.

    View details for Web of Science ID 000086240300002

    View details for PubMedID 10768837

  • Cytokine fusion constructs as DNA vaccines against tumors. Methods in molecular medicine Maecker, H. T., Syrengelas, A., Levy, R. 2000; 29: 221-239


    Various studies have used DNA vaccination as a method of immunizing against tumors (1-12). As with any tumor vaccine, one challenge is to find a truly tumor-specific antigen (13,14). The majority of immunologically targeted tumor antigens are also expressed on a subset of normal host cells. Examples of such antigens include prostate-specific antigen, and CD20, a B cell marker. Some tumor antigens are specific for activated cells of certain types, such as carcinoembryonic antigen (CEA) or the IL-2 receptor. These are often found on embryonic or fetal cells as well as tumor cells. The carbohydrate antigens of melanomas and the immunoglobulin (Ig) idiotype of B cell lymphomas represent tumor-specific antigens (TSA). Unfortunately, TSA have not been identified in more common malignancies. Furthermore, the antigenic determinants of known TSA may differ between patients; for example, the tumor idiotype (Id) of B cell lymphoma is highly patient-specific and must be determined for each case.

    View details for DOI 10.1385/1-59259-688-6:221

    View details for PubMedID 21374323

Conference Proceedings

  • Disturbed NK Cell Compartment In Human CML and Bcr-Abl Positive Mice. Chen, C. I., Koschmieder, S., Kamp, L., Altvater, B., Pscherer, S., Maecker, H., Berdel, W. E., Juergens, H., Lee, P. P., Rossig, C. AMER SOC HEMATOLOGY. 2010: 518-518
  • Cord blood antigen presenting cells show selective deficits in inflammatory responses Drohan, L., Holm, B., Holmes, T., Tongson, E. C., Dekker, C., Maecker, H., Mellins, E. NATURE PUBLISHING GROUP. 2004: 390A-390A
  • Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry Maecker, H. T., Auffermann-Gretzinger, S., Nomura, L. E., Liso, A., Czerwinski, D. K., Levy, R. AMER ASSOC CANCER RESEARCH. 2001: 902S-908S


    Cytokine flow cytometry (CFC) is a simple and powerful method for measuring antigen-specific T-cell responses by detection of intracellular cytokine staining. We applied this method to the detection of CD4 T-cell responses to tumor vaccines. Patients with multiple myeloma were immunized against their autologous tumor immunoglobulin idiotype, using antigen-pulsed dendritic cell vaccination. Blood samples were drawn before and after vaccination, and CFC and proliferation assays were performed. For CFC, whole blood was incubated overnight with antigen in the presence of costimulatory antibodies to CD28 and CD49d. The blood was then treated with EDTA, erythrocytes were lysed, and leukocytes were fixed, permeabilized, and stained for intracellular cytokines [tumor necrosis factor-alpha (TNF-alpha) or IFN-gamma], CD4, and CD69. Cells were analyzed by flow cytometry and cytokine-producing CD69+ cells enumerated as a percentage of CD4 cells. Of nine patients analyzed, three demonstrated detectable CFC responses to tumor immunoglobulin and/or keyhole limpet hemocyanin (KLH) after vaccination. One of these patients responded only to KLH, whereas the other two responded to both tumor immunoglobulin and KLH. Most responses were detected with both TNF-alpha and IFN-gamma, but one patient's KLH response was detected only with TNF-alpha. There was a positive, but not strong, correlation of cytokine responses with proliferative responses to KLH. Although further follow-up and correlation with clinical outcome is needed, CFC may represent a simple yet detailed assessment of T-cell frequencies and subsets responding to cancer vaccines.

    View details for Web of Science ID 000168016200022

    View details for PubMedID 11300490

  • Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression Asanuma, H., Sharp, M., Maecker, H. T., Maino, V. C., Arvin, A. M. OXFORD UNIV PRESS INC. 2000: 859-866


    Memory T cells specific for varicella-zoster virus (VZV), herpes simplex virus (HSV), and human cytomegalovirus (HCMV) were compared in immune adults by intracellular cytokine (ICC) detection. The mean percentages of CD4+ T cells were 0.11% for VZV and 0.22% for HSV by interferon (IFN)-gamma production; the frequency for HCMV was significantly higher at 1.21%. Percentages of VZV-, HSV-, and HCMV-specific CD4+ T cells were similar by use of tumor necrosis factor (TNF)-alpha. HCMV-stimulated CD8+ T cells produced IFN-gamma (1.11%) and TNF-alpha (1.71%); VZV- and HSV-specific CD8+ T cells were not detectable. VZV CD4+ T cell numbers were similar in young adults with natural or vaccine-induced immunity. VZV CD4+ T cells were significantly less frequent in older adults. Secondary varicella immunization did not increase VZV-specific CD4+ T cell frequencies by ICC assay. Numbers of memory T cells specific for herpesviruses may vary with sites of viral latency and with host age.

    View details for Web of Science ID 000086344400007

    View details for PubMedID 10720505