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ABSTRACT: Systems-level approaches are increasingly common in both murine and human translational studies. These approaches employ multiple high information content assays. As a result, there is a need for tools to integrate heterogeneous types of laboratory and clinical/demographic data, and to allow the exploration of that data by aggregating and/or segregating results based on particular variables (e.g., mean cytokine levels by age and gender).
Here we describe the application of standard data warehousing tools to create a novel environment for user-driven upload, integration, and exploration of heterogeneous data. The system presented here currently supports flow cytometry and immunoassays performed in the Stanford Human Immune Monitoring Center, but could be applied more generally.
Users upload assay results contained in platform-specific spreadsheets of a defined format, and clinical and demographic data in spreadsheets of flexible format. Users then map sample IDs to connect the assay results with the metadata. An OLAP (on-line analytical processing) data exploration interface allows filtering and display of various dimensions (e.g., Luminex analytes in rows, treatment group in columns, filtered on a particular study). Statistics such as mean, median, and N can be displayed. The views can be expanded or contracted to aggregate or segregate data at various levels. Individual-level data is accessible with a single click. The result is a user-driven system that permits data integration and exploration in a variety of settings. We show how the system can be used to find gender-specific differences in serum cytokine levels, and compare them across experiments and assay types.
We have used the tools and techniques of data warehousing, including open-source business intelligence software, to support investigator-driven data integration and mining of diverse immunological data.
Journal of Translational Medicine 03/2012; 10:62. · 3.41 Impact Factor
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ABSTRACT: The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.
Nature Reviews Immunology 01/2012; 12(3):191-200. · 32.25 Impact Factor
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Nature Reviews Immunology 01/2012; 12(5):396. · 32.25 Impact Factor
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Lisa H Butterfield,
A Karolina Palucka,
Cedrik M Britten,
Madhav V Dhodapkar,
Leif Håkansson,
Sylvia Janetzki,
Yutaka Kawakami,
Thomas-Oliver Kleen,
Peter P Lee,
Cristina Maccalli, [......],
Hideaki Tahara,
Magdalena Thurin,
Giorgio Trinchieri,
Sjoerd H van Der Burg,
Theresa L Whiteside,
Jon M Wigginton,
Francesco Marincola,
Samir Khleif,
Bernard A Fox,
Mary L Disis
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ABSTRACT: 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.
Clinical Cancer Research 05/2011; 17(10):3064-76. · 7.74 Impact Factor
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Nature Immunology 03/2011; 12(3):271. · 26.01 Impact Factor
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ABSTRACT: 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-γ, IL-2 and/or TNF-α 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.
Journal of immunological methods 01/2011; 363(2):143-57. · 2.35 Impact Factor
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Holden T Maecker,
J Philip McCoy,
Michael Amos,
John Elliott,
Adolfas Gaigalas,
Lili Wang,
Richard Aranda,
Jacques Banchereau,
Chris Boshoff,
Jonathan Braun, [......],
Michael Lotze,
Jerry Nepom,
Hans Ochs,
Raegan O'Lone,
Deborah Phippard,
Scott Plevy,
Stephen Rich,
Mario Roederer,
Dan Rotrosen,
Jung-Hua Yeh
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ABSTRACT: 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.
Nature Immunology 11/2010; 11(11):975-8. · 26.01 Impact Factor
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ABSTRACT: 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.
Current opinion in endocrinology, diabetes, and obesity 08/2010; 17(4):322-8.
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ABSTRACT: 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.
Immunity 10/2009; 31(4):527-8. · 21.64 Impact Factor
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ABSTRACT: 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.
Cytometry Part A 07/2008; 73(11):984-91. · 3.73 Impact Factor
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ABSTRACT: 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.
Blood 07/2008; 111(11):5342-9. · 9.90 Impact Factor
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ABSTRACT: 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.
Cytometry Part A 05/2008; 73(4):289-98. · 3.73 Impact Factor
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Sylvia Janetzki,
Katherine S Panageas,
Leah Ben-Porat,
Jean Boyer,
Cedrik M Britten,
Timothy M Clay,
Michael Kalos, Holden T Maecker,
Pedro Romero,
Jianda Yuan,
W Martin Kast,
Axel Hoos
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ABSTRACT: 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.
Cancer Immunology and Immunotherapy 04/2008; 57(3):303-15. · 3.70 Impact Factor
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ABSTRACT: 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.
Clinical Infectious Diseases 03/2008; 46(3):458-66. · 9.15 Impact Factor
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Holden T Maecker,
Jeffrey Hassler,
Janice K Payne,
Amanda Summers,
Karrie Comatas,
Manar Ghanayem,
Michael A Morse,
Timothy M Clay,
Herbert K Lyerly,
Sonny Bhatia,
Smita A Ghanekar,
Vernon C Maino,
Corazon Delarosa,
Mary L Disis
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ABSTRACT: 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.
BMC Immunology 02/2008; 9:9. · 2.53 Impact Factor
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Margaret Inokuma,
Corazon dela Rosa,
Charles Schmitt,
Perry Haaland,
Janet Siebert,
Douglas Petry,
Mengxiang Tang,
Maria A Suni,
Smita A Ghanekar,
Daiva Gladding,
John F Dunne,
Vernon C Maino,
Mary L Disis, Holden T Maecker
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ABSTRACT: 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.
The Journal of Immunology 09/2007; 179(4):2627-33. · 5.79 Impact Factor
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ABSTRACT: 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.
Journal of Immune Based Therapies and Vaccines 02/2007; 5:7.
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Elizabeth Sinclair,
Qi Xuan Tan,
Margaret Sharp,
Valerie Girling,
Chungkee Poon,
Mark Van Natta,
Douglas A Jabs,
Margaret Inokuma, Holden T Maecker,
Barry Bredt,
Mark A Jacobson
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ABSTRACT: 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.
The Journal of Infectious Diseases 12/2006; 194(11):1537-46. · 6.41 Impact Factor
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ABSTRACT: 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.
Cytometry Part A 10/2006; 69(9):1037-42. · 3.73 Impact Factor
