Single-cell Analysis Techniques Research Articles

Label-free biomarkers of human embryonic stem cell differentiation to hepatocytes.

Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting. © 2016 International Society for Advancement of Cytometry.

One-Cell Analysis as a Technique for True Single-Cell Analysis of Organelles in Breast Tumor and Adjacent Normal Tissue to Profile Fatty Acid Composition of Triglyceride Species

Breast cancer develops in an adipose rich environment of normal adipocytes that are known to aid in tumor progression through an unknown method of lipid transfer from normal cells to tumor cells. Much research is built around lipid analysis of breast tumor and adjacent normal tissues to identify variations in the lipidome to gain an understanding of the role lipids play in progressing cancer. Ideally, single-cell analysis methods coupled to mass spectrometry that retain spatial information are best suited for this endeavor. However, many single-cell analysis methods are not capable of subcellular analysis of intact lipids while maintaining spatial information. One-Cell analysis is a true single-cell technique with the precision to extract single organelles from intact tissues while not interfering or disrupting adjacent cells. This method is used to extract and analyze single organelles from individual cells using nanomanipulation coupled to nanoelectrospray ionization mass spectrometry. Presented here is a demonstration of the analysis of single lipid bodies from two different sets of breast tumor and normal adjacent tissues to elucidate the fatty acid composition of triglycerides using One-Cell analysis coupled to tandem mass spectrometry. As a result, thirteen fatty acid species unique to the tumor tissues were identified, five in one set of tissues and eight in the other set.

Single-cell profiling approaches to probing tumor heterogeneity.

Tumor heterogeneity is a major hindrance in cancer classification, diagnosis and treatment. Recent technological advances have begun to reveal the true extent of its heterogeneity. Single-cell analysis (SCA) is emerging as an important approach to detect variations in morphology, genetic or proteomic expression. In this review, we revisit the issue of inter- and intra-tumor heterogeneity, and list various modes of SCA techniques (cell-based, nucleic acid-based, protein-based, metabolite-based and lipid-based) presently used for cancer characterization. We further discuss the advantages of SCA over pooled cell analysis, as well as the limitations of conventional techniques. Emerging trends, such as high-throughput sequencing, are also mentioned as improved means for cancer profiling. Collectively, these applications have the potential for breakthroughs in cancer treatment.

Detection of cross-sex chimerism in the common marmoset monkey (Callithrix jacchus) in interphase cells using fluorescence in situ hybridisation probes specific for the marmoset X and Y chromosomes.

Chimerism associated with placental sharing in marmosets has been traditionally analysed using conventional chromosome staining on metaphase spreads or polymerase chain reaction. However, the former technique requires the presence of proliferating cells, whereas the latter may be associated with possible blood cell contamination. Therefore, we aimed to develop a single-cell analysis technique for sexing marmoset cells. We applied fluorescent in situ hybridisation (FISH) to cell nuclei using differentially labelled X and Y chromosome-specific probes. Herein we present the validation of this method in metaphase cells from a marmoset lymphoblastoid cell line, as well as application of the method for evaluation of cross-sex chimerism in interphase blood lymphocytes and haematopoietic bone marrow cells from marmosets of same- and mixed-sex litters. The results show conclusively that haematopoietic cells of bone marrow and leucocytes from blood are cross-sex chimeric when the litter is mixed sex. In addition, single samples of liver and spleen cell suspensions from one individual were tested. Cross-sex chimerism was observed in the spleen but not in liver cells. We conclude that FISH is the method of choice to identify cross-sex chimerism, especially when combined with morphological identification of nuclei of different cell types, which will allow a targeted tissue-specific analysis.

Statistical Mechanical Framework for Predicting Cellular Responses from Single-Cell Data

Developments in single-cell analysis techniques have provided detailed resolution on cellular component copy number and variation within a cell population. These data provide a probability distribution for all possible states of the cell, as determined by the measured component copy number per cell. We have developed a statistical mechanical framework that uses single-cell protein population data to model the effective interactions of the measured proteins. This method enables us to predict ensemble changes in cellular components under a variety of perturbations. This framework uses Bayesian inference to compare given models or descriptions of the system, as well as determine the uncertainty in the parameter estimations given the data. We have applied this methodology to study the impact of changes in oxygen partial pressure on the behavior of glioblastoma multiforme (GBM) cancer cells. Our framework shows how we can determine the coupling of oxygen concentration changes to the measured proteins and their underlying effective interactions.

Use of CE to Analyze Solutes in Pico- and Nano-Liter Samples from Plant Cells and Rhizosphere.

This chapter describes the use of capillary electrophoresis (CE) in the accurate quantitative mapping of small molecules and ions in intact function tissues between individual cells at single cell resolution. It can also be used for the analysis of the heterogeneity of soil surrounding roots at similar spatial resolution, providing a link between plant and environment. No pretreatment or genetic manipulation of the plant is required. The application is an extension of the Single Cell Sampling and Analysis technique (SiCSA), in which glass micromanipulation of microcapillaries allows samples in the pl and nl volume range to be obtained and manipulated under paraffin oil (to prevent evaporation) before being introduced to the CE column. An advantage of this approach is that the entire sample can be brought to the detector (without the loading losses associated with other techniques). The power of SiCSA-CE is that the results can be directly related to a range of other single-cell resolution parameters ranging from mechanical and hydraulic properties to gene expression. Several protocols and (contrasting) applications are provided.

Single Cell Analysis of IG Genes in CLL: Cases with Multiple IGH Rearrangements Are Constituted of Several Independent Clones Even When Indistinguishable By Flow Cytometry

Abstract Current evidence suggests that in 2-5% of chronic lymphocytic leukemia (CLL) cases, multiple B cell clones - each expressing unique productive immunoglobulin gene rearrangements (P-IGH)- can be detected. However, only in 35% of them, coexistence of at least two leukemic clones was revealed by flow-cytometry (Plevova et al, Haematologica 2014). In the remaining 65% of cases, presence of multiple clones was presumed based on the number of immunoglobulin gene rearrangements detected, but underlying biological cause, also possibly including lack of allelic exclusion, was not satisfactorily proven at the level of single cells. Therefore, we developed a technique for simultaneous analysis of IGH, IGK and IGL rearrangements at the single cell level and performed further investigation of cases with multiple P-IGH. In years 2006-2015, patients with multiple clonal P-IGHs were searched among 1670 CLL patients examined for IGHV mutation status. Identified cases were submitted to flow-cytometric measurement of surface markers CD5, CD19, CD20, CD23, CD43, CD45, sIgK, sIgL and FMC7 on sorted cells where possible. Based on this examination, patients with clonal P-IGHs exceeding the number of CLL populations distinguished by flow-cytometry were tested using single cell analysis. Patients with corresponding number of P-IGHs and CLL populations (i.e. confirmed as biclonal CLL according to the flow-cytometry and PCR-based IGH detection) served as a control of the technique accuracy. Single cell analysis technique was developed at our department to detect transcribed IGH, IGK and IGL simultaneously in individual cells. B cells from peripheral blood of CLL patients were separated by gradient centrifugation with depletion of non-B cells. Single CD19+ cells were then sorted using FACS Aria III into 96-well plates containing lysis buffer, followed by 2 rounds of multiplex nested RT-PCR, capillary electrophoresis and Sanger sequencing. For each patient, 1-3 plates were analyzed. To consider an IG rearrangement clonal, it had to be detected at least in 3 wells. We detected multiple clonal P-IGHs in 76/1670 (4,6%) CLL patients analyzed. Expression of surface markers was assessed by flow cytometry in 37/76 patients: In 24/37(65%), the number of P-IGHs exceeded number of distinguished populations. Single cell analysis was performed in 16/24 cases, 15/16 patients displayed only one homogeneous CLL population by flow cytometry and 1/16 patient displayed two distinguished populations but three P-IGHs. Two patients with corresponding number of P-IGHs and CLL populations were used as a control. The median of wells tested per patient was 92 and P-IGH detection efficacy 83%. In 12/16 tested patients, as well as in two controls, no cell with more than one P-IGH was detected, confirming the expansion of multiple B-cell clones in all of them. Also, based on the structure of detected clonal IGH rearrangements in each case, a possibility of VH replacement was excluded. In 2 cases, we observed intraclonal diversification within one of the present clones (expressing either IGHV1-69, or IGHV3-72), a rare phenomenon described in CLL. In the remaining 4/16 cases we failed to detect one of the expected P-IGHs likely due to its underrepresentation in a sample, which is supported by results obtained from quantitative PCR with allele-specific primers in a bulk sample. Light chains were successfully detected in 10/12 analyzed cases and two controls; in 4 cases, single cell analysis revealed transcribed clonal IG rearrangements previously undetected in bulk samples. Importantly, each identified clonal IGK/IGL was repeatedly detected exclusively with only one distinct clonal P-IGH, thus constituting independent B cell receptors in independent leukemic clones. We confirm and substantially extend the notion that oligoclonality is the major cause of multiple P-IGH detection in CLL. Obtained information on P-IGH and P-IGK/L pairing will help in further investigation of IG receptors in oligoclonal CLL, as the biological background of oligoclonality in CLL still remains to be elucidated. Supported by grants IGA NT13493-4/2012, MUNI/A/1180/2014 and AZV 15-30015A. Disclosures No relevant conflicts of interest to declare.

Numerical Simulations of the Digital Microfluidic Manipulation of Single Microparticles.

Single-cell analysis techniques have been developed as a valuable bioanalytical tool for elucidating cellular heterogeneity at genomic, proteomic, and cellular levels. Cell manipulation is an indispensable process for single-cell analysis. Digital microfluidics (DMF) is an important platform for conducting cell manipulation and single-cell analysis in a high-throughput fashion. However, the manipulation of single cells in DMF has not been quantitatively studied so far. In this article, we investigate the interaction of a single microparticle with a liquid droplet on a flat substrate using numerical simulations. The droplet is driven by capillary force generated from the wettability gradient of the substrate. Considering the Brownian motion of microparticles, we utilize many-body dissipative particle dynamics (MDPD), an off-lattice mesoscopic simulation technique, in this numerical study. The manipulation processes (including pickup, transport, and drop-off) of a single microparticle with a liquid droplet are simulated. Parametric studies are conducted to investigate the effects on the manipulation processes from the droplet size, wettability gradient, wetting properties of the microparticle, and particle-substrate friction coefficients. The numerical results show that the pickup, transport, and drop-off processes can be precisely controlled by these parameters. On the basis of the numerical results, a trap-free delivery of a hydrophobic microparticle to a destination on the substrate is demonstrated in the numerical simulations. The numerical results not only provide a fundamental understanding of interactions among the microparticle, the droplet, and the substrate but also demonstrate a new technique for the trap-free immobilization of single hydrophobic microparticles in the DMF design. Finally, our numerical method also provides a powerful design and optimization tool for the manipulation of microparticles in DMF systems.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis.

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.

Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons.

New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

In situ metabolic analysis of single plant cells by capillary microsampling and electrospray ionization mass spectrometry with ion mobility separation.

Advances in single cell analysis techniques have demonstrated cell-to-cell variability in both homogeneous and heterogeneous cell populations strengthening our understanding of multicellular organisms and individual cell behaviour. However, additional tools are needed for non-targeted metabolic analysis of live single cells in their native environment. Here, we combine capillary microsampling with electrospray ionization (ESI) mass spectrometry (MS) and ion mobility separation (IMS) for the analysis of various single A. thaliana epidermal cell types, including pavement and basal cells, and trichomes. To achieve microsampling of different cell types with distinct morphology, custom-tailored microcapillaries were used to extract the cell contents. To eliminate the isobaric interferences and enhance the ion coverage in single cell analysis, a rapid separation technique, IMS, was introduced that retained ions based on their collision cross sections. For each cell type, the extracted cell material was directly electrosprayed resulting in ∼200 peaks in ESI-MS and ∼400 different ions in ESI-IMS-MS, the latter representing a significantly enhanced coverage. Based on their accurate masses and tandem MS, 23 metabolites and lipids were tentatively identified. Our results indicated that profound metabolic differences existed between the trichome and the other two cell types but differences between pavement and basal cells were hard to discern. The spectra indicated that in all three A. thaliana cell types the phenylpropanoid metabolism pathway had high coverage. In addition, metabolites from the subpathway, sinapic acid ester biosynthesis, were more abundant in single pavement and basal cells, whereas compounds from the kaempferol glycoside biosynthesis pathway were present at significantly higher level in trichomes. Our results demonstrate that capillary microsampling coupled with ESI-IMS-MS captures metabolic differences between A. thaliana epidermal cell types, paving the way for the non-targeted analysis of single plant cells and subcellular compartments.

QSpec: online control and data analysis system for single-cell Raman spectroscopy.

Single-cell phenotyping is critical to the success of biological reductionism. Raman-activated cell sorting (RACS) has shown promise in resolving the dynamics of living cells at the individual level and to uncover population heterogeneities in comparison to established approaches such as fluorescence-activated cell sorting (FACS). Given that the number of single-cells would be massive in any experiment, the power of Raman profiling technique for single-cell analysis would be fully utilized only when coupled with a high-throughput and intelligent process control and data analysis system. In this work, we established QSpec, an automatic system that supports high-throughput Raman-based single-cell phenotyping. Additionally, a single-cell Raman profile database has been established upon which data-mining could be applied to discover the heterogeneity among single-cells under different conditions. To test the effectiveness of this control and data analysis system, a sub-system was also developed to simulate the phenotypes of single-cells as well as the device features.

Biological noise to get a sense of direction: an analogy between chemotaxis and stress response

Biological noise to get a sense of direction: an analogy between chemotaxis and stress response

Frontier microfluidic techniques for short and long-term single cell analysis

Here, we review the frontier microfluidic techniques for single cell analysis (SCA), which is important for research of many biological systems. Microfluidics provides high-throughput, high-resolution experiments at low cost and reagent use, making it especially useful for single cell analysis. Recent advancements in the field have made SCA more feasible, improving device throughput and resolution, adding capabilities, and combining different functions to bring forth new assays. Developments in incubation have allowed for long-term cell tracking assays to be performed with single cell resolution. The ability of systems to provide chemical isolation or prolonged growth of adherent cells is also discussed.

The prevalence and origin of exoprotease-producing cells in the Bacillus subtilis biofilm

Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.

Atomic Force Microscopy: A New Look at Pathogens

Microbial cells are highly complex and heterogeneous systems. In general, cell populations contain subgroups of cells which exhibit differences in growth rate as well as resistance to stress and drug treatment [1]. In addition, individual cells are spatially organized and heterogeneous, and this cellular heterogeneity is used to perform key functions [2]. This complexity emphasizes the need for single-cell analysis techniques in microbial research. Fluorescence imaging is a powerful tool to localize molecules in single cells [3], [4], but the resolution remains limited to the wavelength of the light source. On the other hand, high-resolution images of microbial structures can be obtained by electron microscopy techniques. In particular, cryo-electron tomography—or three-dimensional (3-D) electron microscopy—provides images of whole bacterial cells, at resolutions that are one to two orders of magnitude higher than those obtained with light microscopy [5]. In the past 20 years a new form of microscopy, atomic force microscopy (AFM), has revolutionized the way researchers probe the microbial cell surface. Instead of using an incident beam, AFM measures the minute forces acting between a sharp tip and the sample [6]–[8]. To generate a topographic image, the tip is attached to a cantilever that bends under force and is moved in three-dimensions using a piezoelectric scanner. While scanning the sample surface, cantilever's bending is measured by a laser beam focused on the free end of the cantilever and reflected into a photodiode. Unlike other microscopy techniques, 3-D images of cells and membranes are obtained at high resolution without staining, labelling or fixation, thus in physiological conditions. AFM is much more than a surface-imaging tool in that it also measures the localization and mechanical properties of the individual cell surface molecules. In this modality, known as single-molecule force spectroscopy, the cantilever deflection is recorded as a function of the vertical displacement of the scanner (as the sample is pushed towards the tip and it retracts) [6], [7]. This results in a cantilever deflection vs. scanner displacement curve, which is transformed into a force-distance curve using appropriate corrections. The characteristic adhesion force between tip and sample measured during retraction is used to probe the distribution and mechanics of single molecules, such as cell surface receptors. These novel AFM techniques complement traditional methods used to analyse microbial cell walls and provide new opportunities for understanding cell surface interactions. In this review, we provide a flavour of the various applications offered by AFM in microbiology, and we highlight some of the key breakthroughs the technique has enabled in pathogen research.

Multiparameter single-cell profiling of human CD4+FOXP3+ regulatory T-cell populations in homeostatic conditions and during graft-versus-host disease

AbstractUnderstanding the heterogeneity of human CD4+FOXP3+ regulatory T cells (Tregs) and their potential for lineage reprogramming is of critical importance for moving Treg therapy into the clinics. Using multiparameter single-cell analysis techniques, we explored the heterogeneity and functional diversity of human Tregs in healthy donors and in patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). Human Tregs displayed a level of complexity similar to conventional CD4+ effector T cells with respect to the expression of transcription factors, homing receptors and inflammatory cytokines. Single-cell profiling of the rare Treg producing interleukin-17A or interferon-γ showed an overlap of gene expression signatures of Th17 or Th1 cells and of Tregs. To assess whether Treg homeostasis is affected by an inflammatory and lymphopenic environment, we characterized the Treg compartment in patients early after alloHSCT. This analysis suggested a marked depletion of Treg with a naive phenotype in patients developing acute graft-versus-host disease, compared with tolerant patients. However, single-cell profiling showed that CD4+FOXP3+ T cells maintain the Treg gene expression signature and Treg-suppressive activity was preserved. Our study establishes that heterogeneity at the single-cell level, rather than lineage reprogramming of CD4+FOXP3+ T cells, explains the remarkable complexity and functional diversity of human Tregs.

Automated Capillary Electrophoresis System for Fast Single-Cell Analysis

Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.

Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry

Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria.

Single-cell analysis techniques reveal a striking heterogeneity of human CD4+ T cell subsets

Distinct CD4+ T cell subsets are necessary for efficient immune responses against various pathogens. Among these, Th17 cells play a critical role in mucosal immunity. On the other hand, Th17 cells are pathogenic in several autoimmune disease models. In addition to IL-17A, Th17 cells also produce the IL-10-related cytokine IL-22. However, recent reports have suggested that human IL-22-producing CD4+ T cells may represent a distinct T helper cell subset indicating an unexpected level of heterogeneity of CD4+ T cell “lineages”. The goal of this study was determine the heterogeneity of inflammatory human CD4+ T cell subsets using single-cell techniques. Individual IL-17 or IFN-g-producing cells were isolated using cytokine secretion assays (CSA) and single-cell sorting. Single-cell gene expression profiling was performed by qRT-PCR using BioMark technology. We first determined which human CD4+ T cell populations secrete IL-17, IL-22, and IFN-g. Analysis of cytokine production at the single cell level revealed that only a small proportion of IL-22-producing cells also produced IL-17, whereas the major IL-22-secreting cell populations were a subset of Th1 cells and “Th22” cells that produce neither IL-17 nor IFN-γ. Finally, we identified a small population of CD4+ T cell producing the 3 cytokines. To define the molecular basis for the observed heterogeneity of inflammatory T cell subsets, we isolated individual IL-17A and IFN-g-producing CD4+ T cells using CSA and analyzed the expression of 48 “marker” genes for Th1, Th2, and Th17 subsets. Hierarchical clustering revealed that the gene expression profiles of Th1 and Th17 cells are distinct at the single-cell level. However, we observed large differences in the expression levels and a remarkable heterogeneity of the expression of marker genes for individual Th1 and Th17 cells. Our data reveal a striking heterogeneity of gene expression within inflammatory T cell populations, which may provide an explanation for the diverse functions of CD4+ T cell subsets in orchestrating immune responses.