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Articles by Holden T. Maecker in JoVE

 JoVE Bioengineering

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

1Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University


JoVE 4398

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Other articles by Holden T. Maecker on PubMed

Dynamics of CD4 and CD8 T Cell Responses to Cytomegalovirus in Healthy Human Donors

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

Epigenetic Changes in Tumor Fas Levels Determine Immune Escape and Response to Therapy

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

Increased Brain Size and Glial Cell Number in CD81-null Mice

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

Human CD81 Directly Enhances Th1 and Th2 Cell Activation, but Preferentially Induces Proliferation of Th2 Cells Upon Long-term Stimulation

CD81, a cell-surface protein of the tetraspanin superfamily, has been shown to costimulate T cell activation in murine T cells, and is involved in development of Th2 immune responses in mice.

Analysis of the Frequencies and of the Memory T Cell Phenotypes of Human CD8+ T Cells Specific for Influenza A Viruses

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

Optimal Preparation of Rhesus Macaque Blood for Cytokine Flow Cytometric Analysis

The rhesus macaque is a common substitute for human subjects in many disease models, including simian immunodeficiency virus, the non-human primate equivalent of the human immunodeficiency virus. Monoclonal antibodies and fluorochromes optimized for use in macaques were included in samples examined for immune responses with the use of intracellular cytokine flow cytometry (CFC).

Performance of Plate-based Cytokine Flow Cytometry with Automated Data Analysis

Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates.

TNF-alpha Detection Using a Flow Cytometric Assay to Determine Cellular Responses to Anthrax Vaccine

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

T Cell Immunity to HIV: Defining Parameters of Protection

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

Strong Cell-mediated Immune Responses Are Associated with the Maintenance of Low-level Viremia in Antiretroviral-treated Individuals with Drug-resistant Human Immunodeficiency Virus Type 1

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

Cytokine Flow Cytometry

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

Persistent and Selective Deficiency of CD4+ T Cell Immunity to Cytomegalovirus in Immunocompetent Young Children

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

Cytokine Flow Cytometry: a Multiparametric Approach for Assessing Cellular Immune Responses to Viral Antigens

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

Results of a Cytomegalovirus (CMV)-specific CD8+/interferon- Gamma+ Cytokine Flow Cytometry Assay Correlate with Clinical Evidence of Protective Immunity in Patients with AIDS with CMV Retinitis

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

Analyzing T-cell Responses to Cytomegalovirus by Cytokine Flow Cytometry

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

Selecting Fluorochrome Conjugates for Maximum Sensitivity

Multicolor Flow Cytometric Analysis in SIV-infected Rhesus Macaque

Dysfunction of Simian Immunodeficiency Virus/simian Human Immunodeficiency Virus-induced IL-2 Expression by Central Memory CD4+ T Lymphocytes

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.

Immune Dysfunction and Micrometastases in Women with Breast Cancer

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.

Ex Vivo Analysis of T-cell Function

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.

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).

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.

Maximizing the Retention of Antigen Specific Lymphocyte Function After Cryopreservation

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.

Immune Responses in the Draining Lymph Nodes Against Cancer: Implications for Immunotherapy

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.

IL-2 Production Correlates with Effector Cell Differentiation in HIV-specific CD8+ T Cells

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.

Flow Cytometry Controls, Instrument Setup, and the Determination of Positivity

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.

Protective Immunity to Cytomegalovirus (CMV) Retinitis in AIDS is Associated with CMV-specific T Cells That Express Interferon- Gamma and Interleukin-2 and Have a CD8+ Cell Early Maturational Phenotype

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.

Phenotype and in Vitro Function of Mature MDDC Generated from Cryopreserved PBMC of Cancer Patients Are Equivalent to Those from Healthy Donors

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.

Functional T Cell Responses to Tumor Antigens in Breast Cancer Patients Have a Distinct Phenotype and Cytokine Signature

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.

Results and Harmonization Guidelines from Two Large-scale International Elispot Proficiency Panels Conducted by the Cancer Vaccine Consortium (CVC/SVI)

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.

An Analytical Workflow for Investigating Cytokine Profiles

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.

Poor Predictive Value of Cytomegalovirus (CMV)-specific T Cell Assays for the Development of CMV Retinitis in Patients with AIDS

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).

Development and Dynamics of Robust T-cell Responses to CML Under Imatinib Treatment

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.

Precision and Linearity Targets for Validation of an IFNgamma 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.

Standardization and Optimization of Multiparameter Intracellular Cytokine Staining

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.

Multiparameter Flow Cytometry Monitoring of T Cell Responses

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.

"MIATA"-minimal Information About T Cell Assays

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.

New Technologies for Autoimmune Disease Monitoring

This article will review new technologies used to characterize the immune phenotype of cells and serum for potential use in studies of autoimmunity.

A Model for Harmonizing Flow Cytometry in Clinical Trials

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.

Quality Assurance of Intracellular Cytokine Staining Assays: Analysis of Multiple Rounds of Proficiency Testing

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

Multiparameter Intracellular Cytokine Staining

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.

Recommendations from the ISBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers

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.

Standardizing Immunophenotyping for the Human Immunology Project

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.

The Stanford Data Miner: a Novel Approach for Integrating and Exploring Heterogeneous Immunological Data

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).

New Tools for Classification and Monitoring of Autoimmune Diseases

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.

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