T Cell Immunology
Tools
Immunophenotyping by flow cytometry, a valuable research tool, uses the differential expression of cellular markers to isolate and characterize T cells and their subsets.
Analysis of T-cell subsets using marker profiles
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 (eg, Th1, Th2, Th17, Treg, Th9).
Analysis of human blood
Data shows a 10-color panel run on a 3-laser BD LSRFortessa™ with a blue/red/violet configuration. The panel was used for defining T-cell subsets using lysed whole blood (BD Pharm Lyse™ lysing buffer) from a normal donor. Use of the BD Horizon™ V450 and BD Horizon V500 dyes allows brighter fluorochromes to be used for more dimly expressed antigens, while the use of 10 colors expands the amount of information gained from a single tube.
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 and BD Influx™ 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 regulatory T (Treg) cells by using CD4-enriched cells and sorting on the CD4+CD25int-hiCD127low phenotype, which provides increased efficiency and higher yields.
Tools to take advantage of the power of multicolor flow cytometry
Whether you are just getting started with flow cytometry, designing an advanced multicolor assay, or looking for a simpler way to set up and optimize your instruments, we offer a wealth of tools and information to help you along the way.
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 antibody | Help B cells and switch antibody isotype
production | T cell proliferation and
enhanced 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
| Intracellular pathogens | Parasites | Parasites | Fungi and extracellular bacteria | - | - |
| Harmful Function | Transplant rejection | Autoimmune disease | Allergy, asthma | Allergy | Organ-specific autoimmune
disease | Autoimmune disease | Autoimmune disease,
cancer |
| Extracellular Markers | CD8 | CD4, CXCR3 | CD4, CCR4, Crth2 (human) | CD4 | CD4, CCR6 | CD4, CXCR5 | CD4, CD25 |
| Differentiation Cytokines | - | IFN-γ, 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-γ, TNF, LT-α | IFN-γ, 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 | RORγt, RORα, Stat3 | Bcl-6, MAF | FoxP3, Smad3, Stat5 |
Markers can be altered as a result of cellular environment, differentiation state, and other factors. Key cytokines appear in bold. BD Biosciences offers reagents for molecules that are red.
- Soroosh P, Doherty TA. Th9 and allergic disease. Immunology. 2009;127:450-458.
- Fazilleau N, Mark L, McHeyzer-Williams LJ, McHeyzer-Williams MG. Follicular helper T cells: lineage and location. Immunity. 2009;30:324-335.
