Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments.

MOTIVATION: Cell populations are never truly homogeneous; individual cells exist in biochemical states that define functional differences between them. New technology based on microfluidic arrays combined with multiplexed quantitative polymerase chain reactions now enables high-throughput single-cell gene expression measurement, allowing assessment of cellular heterogeneity. However, few analytic tools have been developed specifically for the statistical and analytical challenges of single-cell quantitative polymerase chain reactions data. RESULTS: We present a statistical framework for the exploration, quality control and analysis of single-cell gene expression data from microfluidic arrays. We assess accuracy and within-sample heterogeneity of single-cell expression and develop quality control criteria to filter unreliable cell measurements. We propose a statistical model accounting for the fact that genes at the single-cell level can be on (and a continuous expression measure is recorded) or dichotomously off (and the recorded expression is zero). Based on this model, we derive a combined likelihood ratio test for differential expression that incorporates both the discrete and continuous components. Using an experiment that examines treatment-specific changes in expression, we show that this combined test is more powerful than either the continuous or dichotomous component in isolation, or a t-test on the zero-inflated data. Although developed for measurements from a specific platform (Fluidigm), these tools are generalizable to other multi-parametric measures over large numbers of events. AVAILABILITY: All results presented here were obtained using the SingleCellAssay R package available on GitHub (http://github.com/RGLab/SingleCellAssay).

Phenotypic and functional profile of HIV-inhibitory CD8 T cells elicited by natural infection and heterologous prime/boost vaccination.

Control of HIV-1 replication following nonsterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8(+) T-cell-mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8(+) T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically defined CD8(+) T-cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8(+) T-cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by recombinant adenovirus type 5 (rAd5) boosting elicited CD8(+) T-cell-mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically infected virus controllers, this activity correlated with HIV-1-specific CD107a or macrophage inflammatory protein 1beta (MIP-1beta) expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8(+) T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8(+) T-cell response that may be optimized in the search for an efficacious HIV-1 vaccine.

Heavy metal contaminants can eliminate quantum dot fluorescence.

Quantum dots (QD) are fluorescent nanocrystals that are highly useful in imaging and flow cytometric analyses. During routine use of monoclonal antibody conjugates of QD, we have occasionally seen partial or total loss of fluorescence when using certain lots of fixative solutions. We hypothesized that a low level contamination with heavy metal cations was responsible, since low level metal contaminants are not uncommon in formalin solutions. By titrating known concentrations of heavy metal cations into staining solutions, we found that millimolar concentrations of ferrous and zinc ions, and as low as 50 nanomolar cupric ions, completely eliminated QD fluorescence. By mass spectroscopic quantification of metals in commercial fixative solutions previously shown to perform poorly or well with regard to QD fluorescence, we confirmed that the presence of copper in solution was correlated with poor performance. Notably, prior addition of EDTA to chelate the divalent cations in these solutions prevented the inhibition of QD fluorescence. Finally, the copper-induced loss of QD fluorescence is irreversible: cells labeled with QD are highly fluorescent and can be rendered nonfluorescent by the addition of cupric sulfate, even after washing extensively. Indeed, these cells can then be successfully stained with other QD reagents, providing a method for immunofluorescence restaining of cells without contaminating fluorescence from the first stain.

Replication-defective adenovirus vectors with multiple deletions do not induce measurable vector-specific T cells in human trials.

The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4(+) T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3, and E4 regions do not induce appreciable expansion of vector-specific CD4(+) T cells.

Virological outcome after structured interruption of antiretroviral therapy for human immunodeficiency virus infection is associated with the functional profile of virus-specific CD8+ T cells.

A clear understanding of the antiviral effects of CD8(+) T cells in the context of chronic human immunodeficiency virus (HIV) infection is critical for the development of prophylactic vaccines and therapeutics designed to support T-cell-mediated immunity. However, defining the potential correlates of effective CD8(+) T-cell immunity has proven difficult; notably, comprehensive analyses have demonstrated that the size and shape of the CD8(+) T-cell response are not necessarily indicative of efficacy determined by measures of plasma viral load. Here, we conducted a detailed quantitative and qualitative analysis of CD8(+) T-cell responses to autologous virus in a cohort of six HIV-infected individuals with a history of structured interruption of antiretroviral therapy (ART) (SIT). The magnitude and breadth of the HIV-specific response did not, by themselves, explain the changes observed in plasma virus levels after the cessation of ART. Furthermore, mutational escape from targeted epitopes could not account for the differential virological outcomes in this cohort. However, the functionality of HIV-specific CD8(+) T-cell populations upon antigen encounter, determined by the simultaneous and independent measurement of five CD8(+) T-cell functions (degranulation and gamma interferon, macrophage inflammatory protein 1beta, tumor necrosis factor alpha, and interleukin-2 levels) reflected the emergent level of plasma virus, with multiple functions being elicited in those individuals with lower levels of viremia after SIT. These data show that the quality of the HIV-specific CD8(+) T-cell response, rather than the quantity, is associated with the dynamics of viral replication in the absence of ART and suggest that the effects of SIT can be assessed by measuring the functional profile of HIV-specific CD8(+) T cells.

Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs.

BACKGROUND: Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. METHODS AND FINDINGS: To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10) particles, two logs lower than that used previously. CONCLUSIONS: Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10) rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.

Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.

Membrane-damaged cells caused by either mechanical trauma or through normal biological processes can produce artifacts in immunophenotyping analysis by flow cytometry. Dead cells can nonspecifically bind monoclonal antibody conjugates, potentially leading to erroneous conclusions, particularly when cell frequencies are low. To date, DNA intercalating dyes (Ethidium monoazaide (EMA), Propidium Iodide, 7AAD, etc.) or Annexin V have been commonly used to exclude dead cells; however, each suffer from technical problems. The first issue with such dyes is the dependence on a consistent dead cell source for fluorescence compensation. Another major issue is the stability of the staining; except for EMA, fixation and permeablization used for intracellular staining procedures can cause loss of fluorescence. EMA requires a UV exposure to covalently bond to DNA; while this dye is effective and is not affected by intracellular treatments it is weakly fluorescent. Here we report on the optimization of a new class of viability dyes, the amine reactive viability dyes (ViD) as a dead cell exclusion marker. The inclusion of ViD into the staining panel was found to be simple, reproducible and can have a significant benefit on the accuracy of identifying appropriate cell populations. We show the fluorescence of cells stained with these dyes correlates with traditional dead cell discriminating markers, even after fixation and permeabilization. Amine reactive viability dyes are a powerful tool for fluorescence immunophenotyping experiments.

Standardization of cytokine flow cytometry assays.

BACKGROUND: 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). RESULTS: 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. CONCLUSION: 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.

Highly multiplexed quantitation of gene expression on single cells.

Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a "bulk" approach that measures expression patterns from 100 cells at a time in high throughput to define gene signatures, and a single-cell approach to define the coordinate expression patterns of multiple genes and reveal unique subsets of cells.

Amine-reactive dyes for dead cell discrimination in fixed samples.

Amine-reactive dyes, also known as LIVE/DEAD fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining. Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). Since amine-reactive dyes are fluorescent when excited by lasers, dead cells can be identified by flow cytometry. This unit describes procedures, troubleshooting, and outcomes for using the two most commonly used amine-reactive dyes, ViViD and Aqua Blue.

Priming immunization with DNA augments immunogenicity of recombinant adenoviral vectors for both HIV-1 specific antibody and T-cell responses.

BACKGROUND: Induction of HIV-1-specific T-cell responses relevant to diverse subtypes is a major goal of HIV vaccine development. Prime-boost regimens using heterologous gene-based vaccine vectors have induced potent, polyfunctional T cell responses in preclinical studies. METHODS: The first opportunity to evaluate the immunogenicity of DNA priming followed by recombinant adenovirus serotype 5 (rAd5) boosting was as open-label rollover trials in subjects who had been enrolled in prior studies of HIV-1 specific DNA vaccines. All subjects underwent apheresis before and after rAd5 boosting to characterize in depth the T cell and antibody response induced by the heterologous DNA/rAd5 prime-boost combination. RESULTS: rAd5 boosting was well-tolerated with no serious adverse events. Compared to DNA or rAd5 vaccine alone, sequential DNA/rAd5 administration induced 7-fold higher magnitude Env-biased HIV-1-specific CD8(+) T-cell responses and 100-fold greater antibody titers measured by ELISA. There was no significant neutralizing antibody activity against primary isolates. Vaccine-elicited CD4(+) and CD8(+) T-cells expressed multiple functions and were predominantly long-term (CD127(+)) central or effector memory T cells and that persisted in blood for >6 months. Epitopes mapped in Gag and Env demonstrated partial cross-clade recognition. CONCLUSION: Heterologous prime-boost using vector-based gene delivery of vaccine antigens is a potent immunization strategy for inducing both antibody and T-cell responses. TRIAL REGISTRATION: ClinicalTrials.gov NCT00102089, NCT00108654.

Intracellular cytokine optimization and standard operating procedure.

We describe here a method for optimizing the use of polychromatic flow cytometry (with up to 17 fluorochromes simultaneously) in surface and intracellular staining of human T lymphocytes. We will highlight and discuss how to procedurally optimize key steps in the experimental process before an intracellular cytokine staining assay protocol is finalized. These include but are not limited to the titration of monoclonal antibodies, use of a dead-cell discriminator and 'dump' channel, selection of a cytokine secretion inhibitor, selection of fixation and permeabilization reagents, and inclusion of compensation controls. Building on this basic protocol, we then establish a polychromatic assay designed to detect five separate functions of T lymphocytes (production of three cytokines and one chemokine, and degranulation) while simultaneously identifying multiple surface markers on the responding cells.

Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine.

BACKGROUND: Gene-based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine. METHODS: VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle-free intramuscular injection. Humoral responses (measured by enzyme-linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme-linked immunospot assay and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured. RESULTS: The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4(+) and CD8(+) T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine-induced HIV-specific T cells evolved over time, with a diminished frequency of interferon- gamma -producing T cells and an increased frequency of interleukin-2-producing T cells at 1 year. CONCLUSIONS: DNA vaccination induced antibody to and T cell responses against 3 major HIV-1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.