Director, Human Immune Monitoring Center (2009 - Present)
Director, Service Centers and Enabling Technologies, Stanford University School of Medicine (2014 - Present)
PhD, Stanford University, Cancer Biology (1988)
BS, Purdue University, Microbiology (1984)
Community and International Work
Human Immunophenotyping Consortium
Standardization of human immune monitoring
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.
- Introduction to Applied Computational Tools in Immunology
IMMUNOL 206 (Win)
Independent Studies (9)
- Directed Reading in Immunology
IMMUNOL 299 (Win, Spr)
- Directed Reading in Microbiology and Immunology
MI 299 (Aut, Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Win, Spr)
- Graduate Research
IMMUNOL 399 (Win, Spr, Sum)
- Graduate Research
MI 399 (Aut, Win, Spr)
- Medical Scholars Research
MI 370 (Aut, Win, Spr)
- Teaching in Immunology
IMMUNOL 290 (Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Win, Spr)
- Undergraduate Research
MI 199 (Aut, Win, Spr)
- Directed Reading in Immunology
Prior Year Courses
- Introduction to Applied Computational Tools in Immunology
IMMUNOL 206 (Win)
- Introduction to Applied Computational Tools in Immunology
IMMUNOL 206 (Spr)
- Seminars in Computational and Systems Immunology
IMMUNOL 310 (Win)
- Seminars in Computational and Systems Immunology
IMMUNOL 310 (Aut, Win, Spr)
- Introduction to Applied Computational Tools in Immunology
Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals.
2016; 12 (10)
Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.
View details for DOI 10.1371/journal.ppat.1005892
View details for PubMedID 27764254
Immune Profiles to Predict Response to Desensitization Therapy in Highly HLA-Sensitized Kidney Transplant Candidates
2016; 11 (4)
Kidney transplantation is the most effective treatment for end-stage kidney disease. Sensitization, the formation of human leukocyte antigen (HLA) antibodies, remains a major barrier to successful kidney transplantation. Despite the implementation of desensitization strategies, many candidates fail to respond. Current progress is hindered by the lack of biomarkers to predict response and to guide therapy. Our objective was to determine whether differences in immune and gene profiles may help identify which candidates will respond to desensitization therapy.Single-cell mass cytometry by time-of-flight (CyTOF) phenotyping, gene arrays, and phosphoepitope flow cytometry were performed in a study of 20 highly sensitized kidney transplant candidates undergoing desensitization therapy. Responders to desensitization therapy were defined as 5% or greater decrease in cumulative calculated panel reactive antibody (cPRA) levels, and non-responders had 0% decrease in cPRA. Using a decision tree analysis, we found that a combination of transitional B cell and regulatory T cell (Treg) frequencies at baseline before initiation of desensitization therapy could distinguish responders from non-responders. Using a support vector machine (SVM) and longitudinal data, TRAF3IP3 transcripts and HLA-DR-CD38+CD4+ T cells could also distinguish responders from non-responders. Combining all assays in a multivariate analysis and elastic net regression model with 72 analytes, we identified seven that were highly interrelated and eleven that predicted response to desensitization therapy.Measuring baseline and longitudinal immune and gene profiles could provide a useful strategy to distinguish responders from non-responders to desensitization therapy. This study presents the integration of novel translational studies including CyTOF immunophenotyping in a multivariate analysis model that has potential applications to predict response to desensitization, select candidates, and personalize medicine to ultimately improve overall outcomes in highly sensitized kidney transplant candidates.
View details for DOI 10.1371/journal.pone.0153355
View details for Web of Science ID 000374131700042
View details for PubMedID 27078882
Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets.
Proceedings of the National Academy of Sciences of the United States of America
2016; 113 (9): E1286-95
Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional "roadmap" of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an "anergic" Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.
View details for DOI 10.1073/pnas.1520180113
View details for PubMedID 26811452
Platinum-conjugated antibodies for application in mass cytometry.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2016; 89 (3): 292-300
Mass cytometry has overcome limitations of fluorescent single cell cytometry by allowing for the measurement of up to currently ∼40 different parameters on a single cell level. However, the cellular proteome comprises many more potential analytes, and current mass cytometry instrumentation allows for theoretically up to 121 different mass detection channels. The labeling of specific probes with appropriate metal ions is a significant hurdle for exploiting more of mass cytometry's analytical capacity. To this end, we here describe the labeling of antibody with natural abundance or isotopically purified platinum as formulated in cisplatin and circumventing the use of chelator-loaded polymers. We confirm the utility of cisplatin-antibody-conjugates for surface, intracellular, and phosphoepitope-specific immunophenotyping, as well as for application in cell surface CD45-based barcoding. Cisplatin-labeling of antibody increases the analytical capacity of the CyTOF(®) platform by two channels based on available reagents, and has the potential to add a total of six channels for detection of specific probes, thus helping to better extend the analytical mass range of mass cytometers. © 2015 International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.22778
View details for PubMedID 26355391
Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium
Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.
View details for DOI 10.1038/srep20686
View details for Web of Science ID 000369825500001
View details for PubMedID 26861911
Immunodynamics: a cancer immunotherapy trials network review of immune monitoring in immuno-oncology clinical trials.
Journal for immunotherapy of cancer
2016; 4: 15-?
The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapy as a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators review the immunodynamic effects of specific classes of immunotherapeutic agents to focus immune assessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.
View details for DOI 10.1186/s40425-016-0118-0
View details for PubMedID 26981245
Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction.
Journal of the American Society of Hypertension
2015; 9 (12): 975-984 e3
There is strong evidence that inflammatory mediators play a key role in the progression to heart failure in patients with systemic hypertension (HTN). The present study aimed to identify a set of cytokines that are associated with early left ventricular (LV) remodeling and dysfunction as captured by echocardiography in patients with HTN in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome. We identified three groups of participants from the cohort: normotensive subjects (normotension; n = 30), HTN with normal LV structure and function (HTN [LV-]; n = 30), and HTN with evidence of adverse LV remodeling (HTN [LV+]; n = 50). We measured cytokines using a 63-plex Luminex platform. Using partial least squares-discriminant analysis, we constructed three latent variables from the measured cytokines that explained 35%-45% of the variance between groups. We identified five common cytokines (interleukin 18, monokine induced by gamma interferon, hepatocyte growth factor, epithelial neutrophil-activating peptide 78, and vascular endothelial growth factor D) with a stable signal which had a major impact on the construction of the latent variables. Among these cytokines, after adjustment for confounders, interleukin 18 remained significantly different between HTN participants with and without LV involvement (P = .02). Moreover, granulocyte-macrophage colony-stimulating factor and leptin showed a consistent upward trend in all HTN patients compared with normotensive subjects. In conclusion, in HTN patients with LV remodeling or/and dysfunction, we identified a set of cytokines strongly associated with LV maladaptation. We also found a distinct profile of inflammatory biomarkers that characterize HTN.
View details for DOI 10.1016/j.jash.2015.10.003
View details for PubMedID 26565110
Algorithmic Tools for Mining High-Dimensional Cytometry Data
JOURNAL OF IMMUNOLOGY
2015; 195 (3): 773-779
The advent of mass cytometry has led to an unprecedented increase in the number of analytes measured in individual cells, thereby increasing the complexity and information content of cytometric data. Although this technology is ideally suited to the detailed examination of the immune system, the applicability of the different methods for analyzing such complex data is less clear. Conventional data analysis by manual gating of cells in biaxial dot plots is often subjective, time consuming, and neglectful of much of the information contained in a highly dimensional cytometric dataset. Algorithmic data mining has the promise to eliminate these concerns, and several such tools have been applied recently to mass cytometry data. We review computational data mining tools that have been used to analyze mass cytometry data, outline their differences, and comment on their strengths and limitations. This review will help immunologists to identify suitable algorithmic tools for their particular projects.
View details for DOI 10.4049/jimmunol.1500633
View details for Web of Science ID 000358070400004
View details for PubMedID 26188071
- Large-Scale and Comprehensive Immune Profiling and Functional Analysis of Normal Human Aging PLOS ONE 2015; 10 (7)
Immunologic Network and Response to Intramyocardial CD34(+) Stem Cell Therapy in Patients With Dilated Cardiomyopathy
JOURNAL OF CARDIAC FAILURE
2015; 21 (7): 572-582
Although stem cell therapy (SCT) is emerging as a potential treatment for patients with dilated cardiomyopathy (DCM), clinical response remains variable. Our objective was to determine whether baseline differences in circulating immunologic and nonimmunologic biomarkers may help to identify patients more likely to respond to intramyocardial injection of CD34(+)-based SCT.We enrolled from January 3, 2011 to March 5, 2012 37 patients with longstanding DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral CD34(+) stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and collection by means of apheresis. CD34(+) cells were labeled with (99m)Tc-hexamethylpropyleneamine oxime to allow assessment of stem cell retention at 18 hours. Response to SCT was predefined as an increase in LVEF of ≥5% at 3 months. The majority (84%) of patients were male with an overall mean LVEF of 27 ± 7% and a median N-terminal pro-B-type natriuretic peptide (NT-proBNP) level of 2,774 pg/mL. Nineteen patients (51%) were responders to SCT. There was no significant difference between responders and nonresponders regarding to age, sex, baseline LVEF, NT-proBNP levels, or 6-minute walking distance. With the use of a partial least squares (PLS) predictive model, we identified 9 baseline factors that were associated with both stem cell response and stem cell retention (mechanistic validation). Among the baseline factors positively associated with both clinical response and stem cell retention were G-CSF, SDF-1, LIF, MCP-1, and MCP-3. Among baseline factors negatively associated with both clinical response and retention were IL-12p70, FASL, ICAM-1, and GGT. A decrease in G-CSF at 3-month follow-up was also observed in responders compared with nonresponders (P = .02).If further validated, baseline immunologic and nonimmunologic biomarkers may help to identify patients with DCM who are more likely to respond to CD34(+)-based SCT.
View details for DOI 10.1016/j.cardfail.2015.03.011
View details for Web of Science ID 000358105900007
View details for PubMedID 25863169
- Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy ARTHRITIS RESEARCH & THERAPY 2015; 17
flowCL: ontology-based cell population labelling in flow cytometry
2015; 31 (8): 1337-1339
Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources.We developed flowCL, a software package that performs semantic labelling of cell populations based on their surface markers and applied it to labelling of the Federation of Clinical Immunology Societies Human Immunology Project Consortium lyoplate populations as a use case.By providing automated labelling of cell populations based on their immunophenotype, flowCL allows for unambiguous and reproducible identification of standardized cell types.Code, R script and documentation are available under the Artistic 2.0 license through Bioconductor (http://www.bioconductor.org/packages/devel/bioc/html/flowCL.html).email@example.comSupplementary data are available at Bioinformatics online.
View details for DOI 10.1093/bioinformatics/btu807
View details for Web of Science ID 000354453700035
View details for PubMedID 25481008
Cytomegalovirus infection enhances the immune response to influenza.
Science translational medicine
2015; 7 (281): 281ra43-?
Cytomegalovirus (CMV) is a β-herpesvirus present in a latent form in most people worldwide. In immunosuppressed individuals, CMV can reactivate and cause serious clinical complications, but the effect of the latent state on healthy people remains elusive. We undertook a systems approach to understand the differences between seropositive and negative subjects and measured hundreds of immune system components from blood samples including cytokines and chemokines, immune cell phenotyping, gene expression, ex vivo cell responses to cytokine stimuli, and the antibody response to seasonal influenza vaccination. As expected, we found decreased responses to vaccination and an overall down-regulation of immune components in aged individuals regardless of CMV status. In contrast, CMV-seropositive young adults exhibited enhanced antibody responses to influenza vaccination, increased CD8(+) T cell sensitivity, and elevated levels of circulating interferon-γ compared to seronegative individuals. Experiments with young mice infected with murine CMV also showed significant protection from an influenza virus challenge compared with uninfected animals, although this effect declined with time. These data show that CMV and its murine equivalent can have a beneficial effect on the immune response of young, healthy individuals, which may explain the ubiquity of CMV infection in humans and many other species.
View details for DOI 10.1126/scitranslmed.aaa2293
View details for PubMedID 25834109
- Cytomegalovirus infection enhances the immune response to influenza SCIENCE TRANSLATIONAL MEDICINE 2015; 7 (281)
Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.
Journal of immunology
2015; 194 (4): 2022-2031
Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.
View details for DOI 10.4049/jimmunol.1402661
View details for PubMedID 25609839
Variation in the Human Immune System Is Largely Driven by Non-Heritable Influences
2015; 160 (1-2): 37-47
There is considerable heterogeneity in immunological parameters between individuals, but its sources are largely unknown. To assess the relative contribution of heritable versus non-heritable factors, we have performed a systems-level analysis of 210 healthy twins between 8 and 82 years of age. We measured 204 different parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated (>50% of variance) and 58% almost completely determined (>80% of variance) by non-heritable influences. In addition, some of these parameters become more variable with age, suggesting the cumulative influence of environmental exposure. Similarly, the serological responses to seasonal influenza vaccination are also determined largely by non-heritable factors, likely due to repeated exposure to different strains. Lastly, in MZ twins discordant for cytomegalovirus infection, more than half of all parameters are affected. These results highlight the largely reactive and adaptive nature of the immune system in healthy individuals.
View details for DOI 10.1016/j.cell.2014.12.020
View details for Web of Science ID 000347923200007
Large-Scale and Comprehensive Immune Profiling and Functional Analysis of Normal Human Aging.
2015; 10 (7)
While many age-associated immune changes have been reported, a comprehensive set of metrics of immune aging is lacking. Here we report data from 243 healthy adults aged 40-97, for whom we measured clinical and functional parameters, serum cytokines, cytokines and gene expression in stimulated and unstimulated PBMC, PBMC phenotypes, and cytokine-stimulated pSTAT signaling in whole blood. Although highly heterogeneous across individuals, many of these assays revealed trends by age, sex, and CMV status, to greater or lesser degrees. Age, then sex and CMV status, showed the greatest impact on the immune system, as measured by the percentage of assay readouts with significant differences. An elastic net regression model could optimally predict age with 14 analytes from different assays. This reinforces the importance of multivariate analysis for defining a healthy immune system. These data provide a reference for others measuring immune parameters in older people.
View details for DOI 10.1371/journal.pone.0133627
View details for PubMedID 26197454
Phenotyping of Live Human PBMC using CyTOF™ Mass Cytometry.
2015; 5 (2)
Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOF™ (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.
View details for PubMedID 27390767
Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry.
Methods in molecular biology (Clifton, N.J.)
2015; 1343: 81-95
The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays.
View details for DOI 10.1007/978-1-4939-2963-4_7
View details for PubMedID 26420710
- Immune monitoring technology primer: flow and mass cytometry. Journal for immunotherapy of cancer 2015; 3: 44-?
Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy.
Arthritis research & therapy
2015; 17: 127-?
The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.
View details for DOI 10.1186/s13075-015-0644-z
View details for PubMedID 25981462
In utero arsenic exposure and fetal immune repertoire in a US pregnancy cohort
2014; 155 (2): 188-197
Arsenic has wide-ranging effects on human health and there is evidence that it alters the immune response by influencing CD4+/CD8+ T cell ratios, IL-2 cytokine levels, and the expression of immune-response genes. We investigated the impact of in utero environmental arsenic exposure on immune development and function in newborns participating in a pregnancy cohort in New Hampshire, U.S., where arsenic levels have exceeded the current EPA maximum contaminant level of 10 μg/L. Our results showed that maternal urinary arsenic concentrations were inversely related to absolute total CD45RA+ CD4+ cord blood CD69+ T cell counts (N=116, p=0.04) and positively associated with CD45RA+ CD69- CD294+ cell counts (p=0.01). In placental samples (N=70), higher in utero urinary arsenic concentrations were positively associated with the expression of IL1β (p=0.03). These data provide evidence that relatively low-level arsenic exposure in utero may alter the fetal immune system and lead to immune dysregulation.
View details for DOI 10.1016/j.clim.2014.09.004
View details for Web of Science ID 000346114300004
- The Split Virus Influenza Vaccine rapidly activates immune cells through Fc gamma receptors VACCINE 2014; 32 (45): 5989-5997
Monitoring the immune competence of cancer patients to predict outcome
CANCER IMMUNOLOGY IMMUNOTHERAPY
2014; 63 (7): 713-719
A new era of cancer immunotherapy has brought not only successful cancer vaccines but also immunomodulators, such as those that target checkpoint blockade in order to induce endogenous host immune responses. However, the immune system of cancer patients can be compromised through multiple means, including immune suppression by the tumor and by prior therapies such as chemotherapy and radiation. Therefore, a comprehensive means of assessing patient immunocompetence would seem helpful for determining whether patients are ready to benefit from immunotherapy, and perhaps even which immunotherapy might be most appropriate for them. Unfortunately, there are no standardized tests for immune competence, nor is there agreement on what to measure and what will be predictive of outcome. In this review, we will discuss the technologies and assays that might be most useful for this purpose. We argue for a comprehensive approach that should maximize the chances of developing predictive biomarkers for eventual clinical use.
View details for DOI 10.1007/s00262-014-1521-3
View details for Web of Science ID 000339873300006
View details for PubMedID 24487923
IFN Priming Is Necessary but Not Sufficient To Turn on a Migratory Dendritic Cell Program in Lupus Monocytes
JOURNAL OF IMMUNOLOGY
2014; 192 (12): 5586-5598
Blood monocytes from children with systemic lupus erythematosus (SLE) behave similar to dendritic cells (DCs), and SLE serum induces healthy monocytes to differentiate into DCs in a type I IFN-dependent manner. In this study, we found that these monocytes display significant transcriptional changes, including a prominent IFN signature, compared with healthy controls. Few of those changes, however, explain DC function. Exposure to allogeneic T cells in vitro reprograms SLE monocytes to acquire DC phenotype and function, and this correlates with both IFN-inducible (IP10) and proinflammatory cytokine (IL-1β and IL6) expression. Furthermore, we found that both IFN and SLE serum induce the upregulation of CCR7 transcription in these cells. CCR7 protein expression, however, requires a second signal provided by TLR agonists such as LPS. Thus, SLE serum "primes" a subset of monocytes to readily (<24 h) respond to TLR agonists and acquire migratory DC properties. Our findings might explain how microbial infections exacerbate lupus.
View details for DOI 10.4049/jimmunol.1301319
View details for Web of Science ID 000337172100018
View details for PubMedID 24829414
Effects of serum and plasma matrices on multiplex immunoassays
2014; 58 (2-3): 224-233
Multiplexed fluorescence or electrochemiluminescence immunoassays of soluble cytokines are commonly performed in the context of human serum or plasma, to look for disease biomarkers and to monitor the immune system in a simple and minimally invasive way. These assays provide challenges due to the complexities of the matrix (serum or plasma) and the presence of many cytokines near the limit of detection of the assay. Here, we compare the readout of matched serum and plasma samples, which are generally correlated. However, a subset of cytokines usually have higher levels in serum, and the non-specific background is significantly increased in serum versus plasma. Presumably as a result of this non-specific background, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum. We further show, through spike recovery experiments, that both serum and plasma inhibit the readout of many cytokines, with some variability between donors, but with serum causing greater inhibition than plasma in many cases. Standard diluents from different vendors can partially reverse this inhibition to varying degrees. Dilution of samples can also partly overcome the inhibitory effect of the matrix. We also show that dilution is nonlinear and differentially affects various cytokines. Together, these data argue that (1) plasma is a more sensitive matrix for detecting changes in certain low-abundance cytokines; (2) calculation of concentrations in serum or plasma matrices is inherently inaccurate; and (3) dilution of samples should not be assumed to be linear, i.e., all comparisons need to be made among similarly diluted samples.
View details for DOI 10.1007/s12026-014-8491-6
View details for Web of Science ID 000336333700008
- Vitamin D Deficiency in a Multiethnic Healthy Control Cohort and Altered Immune Response in Vitamin D Deficient European-American Healthy Controls PLOS ONE 2014; 9 (4)
Cytokine and Chemokine Patterns Across 100 Days after Hematopoietic Stem Cell Transplantation in Children
BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION
2014; 20 (3): 361-369
We mapped the cytokine response to hematopoietic stem cell transplantation (HSCT) by assaying 51 cytokines and chemokines each week for 100 days in 51 children receiving allogeneic (n = 44) or autologous HSCT (n = 7). Assay values were reported as mean fluorescence intensity (MFI). Log transformation converted MFI to clinically relevant measures (ie, pg/mL). We searched for potential markers of transplant complications by using mixed treatment by subject analysis of variance. Global cytokine secretion in HSCT recipients was significantly lower than in concurrent control patients (n = 11). Coincident with the nadir in WBC count, the concentration of many cytokines declined further by the second and third week. All analytes (except monokine induced by gamma interferon [MIG]) subsequently rebounded by week 4 (coincident with engraftment and recovery of WBC count) but often still remained well below control levels. Concurrent with the collective nadir of multiple cytokines, monocyte chemoattractant protein 1 (MCP-1), growth-regulated oncogene alpha (GRO-a), and leptin surged during weeks 2 to 4. High levels of leptin persisted throughout the 100 post-transplant days. Also during weeks 2 to 4, hepatocyte growth factor (HGF) and IL-6 surged in children with complications but not in those without complications. The peak in HGF was more pronounced in veno-occlusive disease (VOD). HGF and IL-6 secretion rose at least 2 weeks before the clinical diagnosis of VOD or graft-versus-host disease (GVHD). From week 4 onward in all groups, the MFI of the cytokine resistin increased to 5 to 15 times above concurrent control. HGF has now emerged in 3 or more biomarker discovery efforts for GVHD (and in our population for VOD as well). HGF (with or without IL-6) should be investigated as a potential predictive biomarker of VOD or GVHD. Alternatively, the hyperinflammatory "signature" provided by a multicytokine assay may be predictive.
View details for DOI 10.1016/j.bbmt.2013.11.026
View details for Web of Science ID 000332816700011
Influence of Frequent Infectious Exposures on General and Varicella-Zoster Virus-Specific Immune Responses in Pediatricians
CLINICAL AND VACCINE IMMUNOLOGY
2014; 21 (3): 417-426
Reexposure to viruses is assumed to strengthen humoral and cellular immunity via the secondary immune response. We studied the effects of frequent exposure to viral infectious challenges on immunity. Furthermore, we assessed whether repetitive exposures to varicella-zoster virus (VZV) elicited persistently high immune responses. Blood samples from 11 pediatricians and matched controls were assessed at 3 time points and 1 time point, respectively. Besides the assessment of general immunity by means of measuring T-cell subset percentages, antibody titers and gamma interferon (IFN-γ)/interleukin 2 (IL-2)-producing T-cell percentages against adenovirus type 5 (AdV-5), cytomegalovirus (CMV), tetanus toxin (TT), and VZV were determined. Pediatricians had lower levels of circulating CD4(+)-naive T cells and showed boosting of CD8(+) effector memory T cells. Although no effect on humoral immunity was seen, repetitive exposures to VZV induced persistently higher percentages of IFN-γ-positive T cells against all VZV antigens tested (VZV glycoprotein E [gE], VZV intermediate-early protein 62 [IE62], and VZV IE63) than in controls. T cells directed against latency-associated VZV IE63 benefitted the most from natural exogenous boosting. Although no differences in cellular or humoral immunity were found between the pediatricians and controls for AdV-5 or TT, we did find larger immune responses against CMV antigens in pediatricians. Despite the high infectious burden, we detected a robust and diverse immune system in pediatricians. Repetitive exposures to VZV have been shown to induce a stable increased level of VZV-specific cellular but not humoral immunity. Based on our observations, VZV IE63 can be considered a candidate for a zoster vaccine.
View details for DOI 10.1128/CVI.00818-13
View details for Web of Science ID 000332036900019
View details for PubMedID 24429070
Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3).
journal of allergy and clinical immunology
2014; 133 (2): 500-510
The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance.Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy.In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13).Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells.In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.
View details for DOI 10.1016/j.jaci.2013.12.1037
View details for PubMedID 24636474
- Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3) JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY 2014; 133 (2): 500-?
- Monitoring the immune competence of cancer patients to predict outcome Cancer Immunol Immnother 2014
Vitamin d deficiency in a multiethnic healthy control cohort and altered immune response in vitamin D deficient European-American healthy controls.
2014; 9 (4)
In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African-Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4+ (p = 0.04) and CD8+ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.
View details for DOI 10.1371/journal.pone.0094500
View details for PubMedID 24727903
Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry.
Science translational medicine
2013; 5 (208): 208ra145-?
Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.
View details for DOI 10.1126/scitranslmed.3006702
View details for PubMedID 24154599
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
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
Apoptosis and other immune biomarkers predict influenza vaccine responsiveness.
Molecular systems biology
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
Experimental pain and opioid analgesia in volunteers at high risk for obstructive sleep apnea.
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 μ-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-1β and tumor necrosis factor-α (TNF-α) were associated with an enhanced sensitivity to the opioid analgesic effect (IL-1β, P = 0.0218; TNF-α, 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 potency.Clinicaltrials.gov NCT00672737.
View details for DOI 10.1371/journal.pone.0054807
View details for PubMedID 23382975
Daily cytokine fluctuations, driven by leptin, are associated with fatigue severity in chronic fatigue syndrome: evidence of inflammatory pathology.
Journal of translational medicine
2013; 11: 93-?
Chronic fatigue syndrome (CFS) is a debilitating disorder characterized by persistent fatigue that is not alleviated by rest. The lack of a clearly identified underlying mechanism has hindered the development of effective treatments. Studies have demonstrated elevated levels of inflammatory factors in patients with CFS, but findings are contradictory across studies and no biomarkers have been consistently supported. Single time-point approaches potentially overlook important features of CFS, such as fluctuations in fatigue severity. We have observed that individuals with CFS demonstrate significant day-to-day variability in their fatigue severity.Therefore, to complement previous studies, we implemented a novel longitudinal study design to investigate the role of cytokines in CFS pathophysiology. Ten women meeting the Fukuda diagnostic criteria for CFS and ten healthy age- and body mass index (BMI)-matched women underwent 25 consecutive days of blood draws and self-reporting of symptom severity. A 51-plex cytokine panel via Luminex was performed for each of the 500 serum samples collected. Our primary hypothesis was that daily fatigue severity would be significantly correlated with the inflammatory adipokine leptin, in the women with CFS and not in the healthy control women. As a post-hoc analysis, a machine learning algorithm using all 51 cytokines was implemented to determine whether immune factors could distinguish high from low fatigue days.Self-reported fatigue severity was significantly correlated with leptin levels in six of the participants with CFS and one healthy control, supporting our primary hypothesis. The machine learning algorithm distinguished high from low fatigue days in the CFS group with 78.3% accuracy.Our results support the role of cytokines in the pathophysiology of CFS.
View details for DOI 10.1186/1479-5876-11-93
View details for PubMedID 23570606
- Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2012
The phenotypic distribution and functional profile of tuberculin-specific CD4 T-cells characterizes different stages of TB infection.
Cytometry. Part B, Clinical cytometry
2012; 82 (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.21041
View details for PubMedID 22961735
- T Cell Assays and MIATA: The Essential Minimum for Maximum Impact IMMUNITY 2012; 37 (1): 1-2
New tools for classification and monitoring of autoimmune diseases
NATURE REVIEWS RHEUMATOLOGY
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
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
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
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
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
Vitamin D Deficient Healthy Individuals Have Decreased Activated T Cells and Altered Lymphocyte Responses to Cytokine Stimulation
WILEY-BLACKWELL. 2011: S19-S19
View details for Web of Science ID 000297621500055
Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers
CLINICAL CANCER RESEARCH
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
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
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
Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond
CANCER IMMUNOLOGY IMMUNOTHERAPY
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
Multiparameter intracellular cytokine staining.
Methods in molecular biology (Clifton, N.J.)
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
Disturbed NK Cell Compartment In Human CML and Bcr-Abl Positive Mice.
AMER SOC HEMATOLOGY. 2010: 518-518
View details for Web of Science ID 000289662201310
A model for harmonizing flow cytometry in clinical trials.
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
- A model for harmonizing flow cytometry in clinical trials NATURE IMMUNOLOGY 2010; 11 (11): 975-978
New technologies for autoimmune disease monitoring
CURRENT OPINION IN ENDOCRINOLOGY DIABETES AND OBESITY
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
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
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.)
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
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 : the journal of the International Society for Analytical Cytology
2008; 73 (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 PubMedID 18612990
Development and dynamics of robust T-cell responses to CML under imatinib treatment patients
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 : the journal of the International Society for Analytical Cytology
2008; 73 (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 PubMedID 18163472
Precision and linearity targets for validation of an IFN gamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides
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
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
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
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
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
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
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
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
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 : the journal of the International Society for Analytical Cytology
2006; 69 (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 PubMedID 16888771
Immune responses in the draining lymph nodes against cancer: Implications for immunotherapy
CANCER AND METASTASIS REVIEWS
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
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
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
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
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
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
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
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