T lymphocytes have been at the forefront of research in the past decade due to our increasing understanding of their role in the clearance of pathogens, their application in immunotherapy, and the association of their dysregulation with a variety of diseases. A key example is seen with the depletion of T cells, mediated by infections such as from human immunodeficiency virus (HIV) and the severe secondary immune deficiencies that develop, such as acquired immune deficiency syndrome (AIDS).1 In healthy humans, mature T cells and T regulatory cells (Tregs) help in maintaining peripheral immune tolerance, preventing autoimmunity against self antigens. Of recent interest have been immune checkpoint inhibitors targeting T cell receptors, such as PD-1 and CTLA-4, which have been successful in un-inhibiting T cell activation and proliferation of antigen-experienced T cells in the tumor microenvironment. This strategy has been used successfully in treatment against melanoma and other tumors.2 Chimeric antigen receptor (CAR)-T cells, such as anti-CD19 CAR-T cells, have been extremely successful (approximately 93% response) against B cell malignancies, such as acute lymphoblastic leukemia.2 Flow cytometry provides a powerful tool for immunophenotyping and analyzing T cells, furthering insights in each of these fields.
BD Biosciences provides a comprehensive portfolio of reagents and kits to study T cells
Among the methods used for studying T cells, multicolor flow cytometry is preeminent because it enables the characterization of highly complex T cell subpopulations—both functionally and phenotypically. Complementary technologies such as ELISA, ELISPOT and bead-based immunoassays also further research in the T cell area, offering flexibility to meet a range of experimental needs and multiple methods to verify results.
The following table lists important characteristics of tools and technologies to support your T cell research and help you find the ones that meet your experimental needs. Certain technologies can reveal specific information about a sample or might better meet practical needs such as the available instrumentation or sample type. In some cases, researchers can use the combined information from multiple techniques to verify results. Different approaches can paint a detailed picture of the mechanisms contributing to T cell development.
|Tool/Technology||Flow Cytometry: Surface||Flow Cytometry: Introcellular||BD® Cytometric Bead Array (CBA)||ELISPOT||ELISA||In Vivo Capture Assay|
|Molecules detected||Surface||Intracellular and surface||Secreted or intracellular||Secreted (in situ)||Secreted||Secreted (in vivo)|
|Single cell/cell subset information||Yes||Yes||No||Frequencies, no subset information||No||No|
|Post-assay viability||Yes||No||Yes, for secreted molecules||No||Yes||Yes|
|Quantitation of protein||Possible*||Possible*||Yes||No||Yes||Yes|
|Instrumentation||Flow cytometer||Flow cytometer||Flow cytometer||ELISPOT reader||Spectrophotometer||Spectrophotometer|
*With a standard such as BD Quantibrite™ Beads
Immunophenotyping: combining surface and intracellular markers
Using multicolor flow cytometry—and combining surface phenotyping with intracellular staining of cytokines, transcription factors or signaling molecules—you can identify individual T cell subsets based on their characteristic marker signature. BD advanced reagent systems, extensive marker selection and wealth of support tools help researchers simplify phenotyping and maximize the information obtained from individual samples.
Immunophenotyping by flow cytometry, a valuable research tool, uses the differential expression of cellular markers to isolate and characterize T cells and their subsets. T cells and their subsets can be defined by differential expression of cell surface markers including CD3, CD4, CD8 and CD25. Using panels of directly conjugated fluorescent antibodies to these specific markers, multicolor flow cytometric analysis allows researchers to interrogate the levels of multiple markers simultaneously on individual cells. This can provide information about the cell lineage and state of differentiation of cell subsets in a particular sample. Adding markers such as CCR7, CD62L or CD69 to an analysis provides important information about the potential for cells to home and localize within the body, as well as the activation status of the T cell subset of interest. By analyzing the expressed patterns of various markers—including not only cell surface receptors but also cytokine secretion profiles and intracellular signaling molecules—researchers have defined phenotypes that represent functionally distinct T cell subsets (e.g., Th1, Th2, Th17, Treg, Th9).
Major Known T Cell Markers
|Type of Cell||Cytotoxic||Th1||Th2||Th91||Th17||Tfh2||Treg|
|Main Function||Kill virus-infected cells||Activate microbicidal function of infected macrophages and help B cells to produce antiboday||Help B cells and switch antibody isotope production||T cell proliferation and enhance IgG and IgE production by B cells||Enhance neutrophil response||Regulate development of antigen specific B cell development and antibody production||Immune regulation|
|Pathogens Targeted||Viruses and some||Inracelluar pathogens||Parasites||Parasites||Fungi and extracellular bacteria|
|Extracellular Markers||CD8||CD4, CXCR3||CD4, CCR4, Cith2 (human)||CD4||CD4, CCR6||CD4, CXCR5||CD4, CD25|
|Differentiation Cytokines||IFN-y, IL-2, IL-12, IL-18, IL-27||IL-4, IL-2, IL-33||IL-4, TGF-β||TGF-β, IL-6, IL-1, IL-21, IL-23||IL-12, IL-6||TGF-β, IL-12|
|Effector Cytokines||IFN-y, TNF, LT-α||IFN-y, LT-α, TNF||IL-4, IL-5, IL-6, IL-13||IL-9, IL-10||IL-17A, IL-17F, IL-21, IL-22, IL-26, TNF, CCL20||IL-21||TGF-β, IL-10|
|Transcription Factors||T-bet, Stat1, Stat6||GATA3, Stat5, Stat6||GATA3, Smads, Stat6||RORyet, RORα, Stat3||Bci-6, MAF||FoxP3, Smad3, Stat5|
Markers can be altered as a result of cellular environment, differentiation state, and other factors. Key cytokine appear in bold. BD Biosciences offers reagents for molecules that are green.
- Soroosh P. Doherty TA. Th9 and allergic disease. Immunology. 2009; 127(4):450-458. doi: 10.1111/j. 1365-2567.209.03114.x.
- Fazilleau N, Mark L, McHeyzer-Williams LJ, McHeyzer-Williams MG. Follicular helper T cells: lineage and location. Immunity. 2009;30(3):324-355. doi: 10.1016/j.immuni.2009.03.003.
View the panel sheet detailing a multicolor flow cytometry panel for the detection of major human circulating to detect CD4+ T cell subsets.
Isolation of selected cell populations by cell sorting
Defined cell populations can be isolated from complex cell mixtures by using a specific combination of surface markers and cell sorters, such as the BD FACSAria™ III, BD FACSMelody™ and BD FACSymphony™ S6 Flow Cytometry Systems. Purified, viable cell populations can be expanded, differentiated and maintained in culture. For certain applications, BD Biosciences scientists have worked out detailed protocols for sorting, such as a method of isolating human Treg cells by using CD4-enriched cells and sorting on the CD4 +CD25 int-hiCD127 low phenotype, which provides increased efficiency and higher yields.
Intracellular cytokine detection for the analysis of T cell subset-specific responses
Detection of intracellular cytokines using multicolor flow cytometry is a powerful tool to gain insight into the spectrum of responses within mixed populations of cells. By simultaneously measuring multiple cytokines and cell surface markers in one assay, this technique offers insight into which cell subset is producing which cytokines. For example, multicolor flow cytometry can be used to identify Th1, Th2 or Th17 cells based on their characteristic cytokine signatures. The cell-by-cell analysis allows researchers to analyze responses of distinct cell subsets without physically separating them.
Intracellular cytokine detection systems
For intracellular protein detection, cells must be fixed and permeabilized to allow a fluorescent antibody to enter and detect the target protein of interest. Different antigens have different sensitivities to and requirements for fixation and permeabilization, entailing additional optimization of protocols. To detect cytokines, which are secreted proteins, protein transport inhibitors are required to trap proteins inside the cells. BD Biosciences offers two widely used assay systems for intracellular cytokine detection: BD Cytofix/Cytoperm™ Reagents and the BD FastImmune™ Cytokine System. Both systems offer researchers the ease and confidence of using tested reagents and protocols and incorporate the high standard of quality and reproducibility that BD flow cytometry products are known for. BD Phosflow™ Reagents help you use multicolor flow cytometry to reliably measure the level of phosphorylated proteins involved in T cell signaling and combine the data with subset identification.
BD Cytofix/Cytoperm™ Reagent method
The BD Cytofix/Cytoperm™ Reagent method and related reagents address the wide-ranging experimental needs of basic research. With products for a broad selection of cytokines and multiple species, BD Cytofix/Cytoperm™ Reagents offer maximum flexibility in intracellular cytokine staining. BD Biosciences offers several solutions for measuring intracellular cytokines through intracellular flow cytometry.
BD FastImmune™ System
The BD FastImmune™ System is designed to meet the needs of applied research on human samples. With a focus on complete systems, this approach is well suited for research studies monitoring immune status during disease or immune responses to vaccine candidates. The BD FastImmune™ CD4 Intracellular Cytokine Detection Kit Anti-Hu-TNF-α/CD69/CD4/CD3 is designed for the detection of intracellular cytokines and the activation marker CD69 in antigen-activated CD4+ T lymphocytes in whole blood. Applications include studies of T cell responses to antigens, such as herpes viruses, HIV and tumor antigens. BD Biosciences offers several solutions for measuring intracellular cytokines through intracellular flow cytometry.
Diagram showing stages of BD FastImmune™ Cytokine System acting on a sample.
BD Phosflow™ Reagent technology offers an extremely powerful means of obtaining kinetic information on T cell subset-specific signaling processes.
T cells are activated and regulated by complex pathways involving several signal transduction molecules, including receptors for antigens and cytokines, kinases, and transcription factors. Signals involving protein phosphorylation play a role in the differentiation of naïve CD4+ T cells into Th1, Th2, Th9, Th17, Tfh or Treg cells.
Detecting transient phosphorylation events: BD Phosflow™ Reagent technology
Innovative BD Phosflow™ Reagent technology was the first complete flow cytometry solution to reveal intracellular data on basal and induced protein phosphorylation events in both cell lines and primary cells. The BD Phosflow™ Reagent approach is especially informative for studying T cells in which phosphorylation of signaling proteins leads to the expression of particular T cell phenotypes.
For detection of secreted cytokines within a sample, BD offers multiple assays. BD® Cytometric Bead Array (CBA) technology allows quantitation of multiple soluble cytokines simultaneously, while BD OptEIA™ ELISA Reagents are designed for quantitation of single cytokines. BD® ELISPOT Reagents enable determination of the frequency of cytokine-producing cells, and BD® In Vivo Capture Assays allow quantitation by directly capturing cytokines in vivo.
The BD Pharmingen™ Human FoxP3 Antibody, available in multiple sizes and conjugates, is a reagent for the detection of FoxP3 positive Tregs. An easy-to-use buffer system allows researchers to fix and permeabilize cells in just a few simple steps, with the option of freezing samples up to 72 hours. While FoxP3 is a commonly used marker for Treg identification, isolation and characterization, Tregs are a very active area of research, and an emerging list of targets has been published in the literature. To support these emerging discoveries, the BD Biosciences portfolio of new high-quality reagents and solutions continues to grow.
CD39: Enhanced characterization of Tregs
Previously localized primarily on B cells, dendritic cells and certain subsets of T cells, CD39 has recently been shown to be co-expressed with FoxP3 in CD4+ Tregs in humans and mice.2 This discovery is adding to the growing list of cell surface markers such as CD25, CD45RA, HLA-DR and CTLA-4, that are important in the identification and functional characterization of CD4+ Tregs.
Extracellular ATP and its metabolites are potent regulatory molecules modulating a broad range of cell and organ functions. Cellular ATP release is an indicator of tissue destruction and a danger signal that activates the immune response. CD39 hydrolyzes extracellular ATP (or other triphosphates) into its respective nucleotides such as AMP. Extracellular nucleoside monophosphates are, in turn, rapidly degraded to nucleosides (e.g., adenosine) by soluble or membrane bound ecto-5′ nucleotidases (CD73). Pericellular adenosine then mediates anti-inflammatory T cell responses. Co-expression of CD39 and CD73 is thought to be one of the key mechanisms of immunosuppression mediated by Tregs.3
Enrichment of Tregs with CD4 and CD25
In humans, initial analysis of Treg populations revealed that only those ex vivo cells that express the highest levels of CD25, which represent approximately 2–3% of total CD4 T cells, demonstrate an in vitro suppressive activity in contrast to mouse cells in which all CD25 cells are considered Tregs. Furthermore, cells expressing low to intermediate levels of CD25 were thought not to exhibit any suppressive activity directly ex vivo.
Additionally, the definition of high and low levels of CD25 expression lacks consensus and has limited its use for obtaining viable human Tregs via flow cytometric cell sorting. As a result, many researchers only select cells with the highest expression of CD25, dramatically reducing the yield of isolated Tregs. These results intensified research to identify cells surface markers other than CD4 and CD25 that are exclusive to human Tregs.4
- Di Caro V, D'Anneo A, Phillips B, et al. Interleukin-7 matures suppressive CD127(+) forkhead box P3 (FoxP3)(+) T cells into CD127(-) CD25(high) FoxP3(+) regulatory T cells. Clin Exp Immunol. 2011;165(1):60-76. doi:10.1111/j.1365-2249.2011.04334.x
- Tøndell A, Wahl SGF, Sponaas AM, Sørhaug S, Børset M, Haug M. Ectonucleotidase CD39 and checkpoint signalling receptor programmed death 1 are highly elevated in intratumoral immune cells in non-small-cell lung cancer. Transl Oncol. 2020;13(1):17-24. doi:10.1016/j.tranon.2019.09.003
- de Oliveira Bravo M, Carvalho JL, Saldanha-Araujo F. Adenosine production: a common path for mesenchymal stem-cell and regulatory T-cell-mediated immunosuppression. Purinergic Signal. 2016;12(4):595-609. doi:10.1007/s11302-016-9529-0
- Attias M, Al-Aubodah T, Piccirillo CA. Mechanisms of human FoxP3+ Treg cell development and function in health and disease. Clin Exp Immunol. 2019;197(1):36-51. doi:10.1111/cei.13290
- Application Notes
- Delineation of Functional T reg Subsets Using Cell Surface Marker Expression - Panel Sheet
- Detection of Intracellular Cytokines in T Lymphocytes using the BD FastImmune™ Assay on the BD FACSVerse™ System
- Studying Mouse Thymocyte Development using Multiparametric Flow Cytometry: An Efficient Method to Improve an 8-Color Panel on the BD FACSVerse™ System
- Frequently Asked Questions
- Product Information Sheets
- BD® CBA Human and Mouse Th1/Th2/Th17 Kit
- BD Pharmingen™ CD4+ T Cell Phenotyping Kits
- BD Pharmingen™ Transcription Factor Buffer Set
- BD Human Regulatory T Cell Cocktail (CD4/CD25/CD127)
- Human CD39 Reagents
- Human FoxP3
- Human Th17 Research Products
- Human and Mouse Reactive T-bet Monoclonal Antibodies
- Mouse Foxp3
- Mouse Th17 Reagents
- Unique Markers for the Study of Human Activated Regulatory T Cells: GARP, LAP, and SATB1
- Activation of Immune Cells for Cytokine Production
- BD Cytofix/Cytoperm Method for Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis
- BD Pharmingen™ Transcription Factor Buffer Set, Cat. Nos. 562725, 562574
- CD3 Plate Stimulation Protocol of Murine T Cells for the Detection of Activation Markers
- Protocol for CD3 Stimulation of Human T Cells for the Detection of Activation Markers
- Simultaneous Measurement of Cell Surface Markers with FoxP3 and Stat5 (pY694) in IL-2– Stimulated Human PBMCs using BD Cytofix™ Fixation Buffer and BD Phosflow™ Perm Buffer III
- Complete, Novel Reagent and Instrumentation Solutions for the Sorting of Viable, Functional Regulatory T Cells
- Optimizing Intracellular Flow Cytometry: Simultaneous Detection of Cytokines and Transcription Factors
- Simplify Immunophenotyping Using Kits & Templates on the BD Accuri™ C6 Flow Cytometer
- Simultaneous Correlation of Cytokine Production with Treg and Th17 Cell Proliferation
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