莱斯特城对富勒姆:T-Cell Research

莱斯特·德·利昂柯特 www.jclehg.com.cn

BD solutions, including flow cytometers, software, conjugated antibodies, and services, are designed to support the rapid pace of discovery in T-cell research. Our solutions for T-cell identification, transcription factor expression, as well as cytokine secretion and measurement, reflect a commitment to high quality and consistency needed for advanced research.

To meet customer requirements,a range of choices is offered, from complete kits for phenotyping and sorting to fully customized formulas.

T cell precursors, derived from hematopoietic stem cells in bone marrow, undergo multiple phases of differentiation in the thymus before maturing into functional T cells.

During this development, precursor T cells transiently or stably express specific molecules including T cell lineage markers such as TCR, CD3, CD4, and CD8. By analyzing expressed patterns of various markers, including cell surface receptors and intracellular signaling molecules, researchers have determined the cellular and molecular mechanisms that enable precursors to develop into mature, functional T cells.


A wide array of applications facilitate these studies—preeminent among them is multicolor flow cytometry. Using panels of directly-conjugated fluorescent antibodies to recognize T cell specific epitopes multicolor flow cytometric analysis allows researchers to interrogate specific target molecule levels expressed by individual cells in various phases of development. Using flow cytometry and assay systems such as BD™ Cytometric Bead Array (CBA) and BD™ Phosflow that measure proteins derived from activated T cell subsets, researchers have gained a detailed view of the cellular pathways and molecular mechanisms that support T cell development.

BD Biosciences also offers products that use complementary technologies such as ELISA, ELISPOT, In Vivo Capture, and intracellular staining assays to support T cell research.

For over 20 years, BD Biosciences has actively supported groundbreaking research in the field, with innovative FACS™ brand flow cytometry systems and high quality BD Pharmingen™ and BD FastImmune™ brand reagents designed to simplify the identification, isolation, and characterization of T cells and their interacting partners.

Helper T cells are essential regulators of adaptive immune responses and inflammatory diseases.

A sub-group of lymphocytes, helper T cells play an important role in establishing and maximizing the capabilities of the immune system. These cells are unusual in that they have little or no cytolytic or phagocytic activity. However, they are involved in activating and directing other immune cells.

After activation by professional antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells, antigen-specific CD4 + T cells differentiate into effector cells that are specialized in terms of the cytokines and effector molecules that they express on their membranes or secrete and their discrete effector functions.


Th1 Cells

Th1 cells are involved in the cellular immune response and host defense against intracellular pathogens. These molecules are characterized by the production of pro-inflammatory cytokines like IFN-γ, IL-2, and lymphotoxin-α (LTα). Th1 cells are centrally involved in cell-mediated immunity. The cytokines produced by Th1 cells stimulate the phagocytosis and destruction of microbial pathogens by macrophages and other lymphocytes. Several chronic inflammatory diseases have been described as Th1-dominant diseases including multiple sclerosis, diabetes, and rheumatoid arthritis.

Th2 Cells

Th2 cells are involved in the humoral immune response and host defense against extracellular parasites. These cells are characterized by the production of IL-4, IL-5, IL-6, IL-10, and IL-13. Th2 cells are thought to play a role in allergic responses. Cytokines like IL-4 generally stimulate the production of antibodies directed toward large extracellular parasites, while IL-5 stimulates eosinophil response toward large extracellular parasites. Allergy and atrophy are thought to be Th2-dominant conditions. Th2 cells have historically been thought to be the source of IL-9. However, recent publications suggest the existence of a Th2-related cell type that is characterized by the secretion of IL-9 and IL-10. These so-called Th9 cells can differentiate from Th2 cells in the presence of TGF-β, or they can differentiate from a naïve CD4 cell with a combination of IL-4 and TGF-β. These cells may be involved in asthma and tissue inflammation.

Th1/Th2 Balance

When Th1 cells produce IFN-γ, this prompts macrophages to produce TNF and toxic forms of oxygen which destroy the microorganisms within the phagosomes and lysosomes. On the other hand, when Th2 cells produce IL-4 and IL-10, these cytokines block the microbial killing that is activated by IFN-γ. The Th1/Th2 relationship has also been investigated in regards to transplantation. Th1 responses have been implicated in most forms of acute graft or transplant rejection and graft-versus-host (GVHD) disease, while Th2 responses have been variably associated with either protection or chronic rejection. However, cloned Th1 or Th2 cells have a similar capacity to reject skin grafts in experimental models, and Tr1/Treg cells are now being implicated in protection and tolerance induction. The fetus is also analogous to an allograft, and Th2 or Treg responses are thought to be protective, while Th1 may lead to resorption or spontaneous abortion.


Th17 cells

Th17 cells

Th17 cells are involved in inflammation and host defense against extracellular pathogens. A subset of helper T cells that produce IL-17A, Th17 cells has been shown to play an important role in the induction of autoimmune tissue injury. They are distinct from Th1 or Th2 cells since they do not produce classical Th1 or Th2 cytokines such as IFN-γ or IL-4. They play a key role in mouse models of autoimmunity, and it has been suggested that the differentiation pathway from a naïve T-helper cell to a Th17 cell involves a combination of TGF-β and IL-6. RORγt is a key transcription factor involved in induction of Th17 cells. Some RORγt expression is induced in response to IL-6 or TGF-β, but the generation of Th17 cells requires TGF-β, as well as IL-6.

Furthermore, it is believed that the relative balance of IL-6 and TGF-β in steady state would tilt the balance in favor of either Th17 or Treg differentiation in diverse tissues. Induction of the Th17 subset requires TGF-β and IL-6, while amplification of IL-17A–producing cells is dependent upon TGF-β and IL-21. Maintenance of a Th17 response primarily depends on IL-23 (p19/p40). IL-23 binds to the IL-23 receptor that triggers downstream activation of STAT3 and subsequent upregulation of ROR-γ and production of IL-17A.

Since IL-17A leads to the induction of many proinflammatory factors such as TNF, IL-6, and IL-1β, it has been suggested that Th17 cells might be responsible for the development and/or progression of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and colitis.

Tools for the identification, isolation, and multicolor analysis of human regulatory T cells

As published data on the immunosuppressive potential of regulatory T cells (Tregs) has accumulated, enthusiasm for their potential therapeutic application has intensified. As a result, Treg research is very active, and new publications emerge almost daily. Today the most commonly used markers for Treg identification, isolation, and characterization are CD4, CD25, CD127, and FoxP3. However, new targets with functional significance such as CD39, CD45RA, CTLA-4, and others are rapidly emerging. 

More on regulatory T cells >>

BD offers a comprehensive portfolio of reagents and kits to study T cells from different perspectives, using a range of technologies.

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.


Comparison of technologies for T cell research

The following table lists important characteristics of tools to help you find the ones that meet your particular 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: Intracellular 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)
Multiparameter Yes Yes Yes No No No
Single cell/cell subset
information
Yes Yes No Frequencies, no subset
information
No No
Antigen-specific Yes Yes Yes Yes Yes Yes
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

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 on the basis of their characteristic marker signature. BD's advanced reagent systems, extensive marker selection, and wealth of support tools help researchers simplify phenotyping and maximize the information obtained from individual samples.

Intracellular cytokines

BD Cytofix/Cytoperm™ reagents and the BD FastImmune™ cytokine system are reagents and protocols optimized to help you use flow cytometry to reproducibly measure cytokines within individual cells.

Intracellular signaling

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.

Secreted cytokines

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.

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 human blood

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).

Major Known T-Cell Markers »

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 +CD25 int-hiCD127 low 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.

Multicolor Flow Cytometry Tools »

 

Major Known T-Cell Markers

Type of Cell Cytotoxic Th1 Th2 Th9 1 Th17 Tfh 2 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.

  1. Soroosh P, Doherty TA. Th9 and allergic disease. Immunology. 2009;127:450-458.
  2. Fazilleau N, Mark L, McHeyzer-Williams LJ, McHeyzer-Williams MG. Follicular helper T cells: lineage and location. Immunity. 2009;30:324-335.

Intracellular cytokine detection allows 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 on the basis of 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 optimized reagents and protocols and incorporate the high standard of quality and reproducibility that BD flow cytometry products are known for.

BD Cytofix/Cytoperm method

The BD Cytofix/Cytoperm 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.

More about BD Cytofix/Cytoperm »

BD FastImmune system

The BD FastImmune system is designed to meet the needs of applied research on human samples. With a focus on complete, optimized systems, this approach is well suited for research studies monitoring immune status during disease, or immune responses to vaccine candidates.

More about BD FastImmune »

BD Phosflow? 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 a number of 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 technology

Innovative BD Phosflow technology is 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 approach is especially informative for studying T cells, in which phosphorylation of signaling proteins leads to the expression of particular T-cell phenotypes.

Specialized antibodies, buffers, and kits

BD provides reagents and kits for the study of protein phosphorylation by flow cytometry. Developed collaboratively by BD and researchers at Stanford University, BD Phosflow technology consists of phosphoprotein-specific, fluorochrome-labeled monoclonal antibodies, along with a system of optimized buffers that fix the cellular proteins to maintain their phosphorylation state and prepare cells for antibody staining.

Researchers can use the BD Phosflow system in combination with cell surface markers to reliably determine the level of key phosphorylated signaling proteins from populations within heterogenous samples such as whole blood. Even rare cell subtypes can be identified without upfront enrichment.

More about BD Phosflow »

Transcription Factor Buffer Set

The BD Pharmingen™ transcription factor buffer set is an optimized fixation and permeabilization buffer system ideal for the detection of intracellular and intranuclear proteins by flow cytometry.

It improves the sensitivity for many transcription factors including, but not limited to, FoxP3, RORγt, T-bet, and Bcl-6.

Features

  • Optimized fixation and permeabilization buffer for the detection of intracellular and intranuclear proteins by flow cytometry
  • Improved sensitivity for many transcription factors including FoxP3, RORγt, Bcl-6, and T-bet
  • Fully integrated novel formulas speed processing and deliver high cell yield
  • Compatible with many cell types, cell surface staining, and tandem dyes
  • Supports high-throughput testing via volume reduction, bulk fixing, and storage

For more information, download the Transcription Factor Buffer Set data sheet »

A Choice of Techniques for Quantification of Secreted Cytokines

Secreted cytokines in samples can be quantified by various techniques. BD™ Cytometric Bead Array (CBA) can simultaneously quantify multiple cytokines from the same sample, while ELISA is a useful assay for measuring levels of single cytokines. BD™ ELISPOT reagents determine the frequency of cytokine-producing cells, while BD™ In Vivo Capture Assays allow sensitive and direct measurement of cytokine production in vivo (in mice)

BD Cytometric Bead Array (CBA)

BD CBA is a bead-based immunoassay for quantifying multiple proteins simultaneously using flow cytometry. The BD CBA portfolio includes assays for the measurement of a variety of soluble and intracellular proteins including cytokines, chemokines, growth factors, and phosphorylated cell signaling proteins. This technology makes BD CBA especially suitable for studying events involved in T-cell differentiation.

More about BD CBA »

BD ELISA reagents

ELISA is a useful assay for quantitating single cytokines. BD offers maximum flexibility, offering a large menu of pre-optimized, ready-to-use BD OptEIA™ kits and sets, as well as individual matched capture and detection antibody pairs with corresponding recombinant protein standards.

More about BD ELISA Reagents »

BD ELISPOT technology

The BD ELISPOT assay is a powerful tool for detecting and analyzing individual cells that secrete a particular protein in vitro. This technology enables determination of the frequency of cytokine-producing cells in a sample. It is highly sensitive and allows for downstream analysis.

More on BD ELISPOT Technology »

BD in vivo capture assays

The BD In Vivo Capture Assay is a complementary technology to ELISA and CBA. It is used for sensitive and direct measurement of cytokine production in vivo (in mice) through the injection of a cytokine-specific antibody and subsequent analysis of collected serum in an ELISA-type assay. The in vivo capture assays are easy to use and yield highly sensitive and directly correlated results.

More on In Vivo Capture Cytokine Assays »

Comparison of technologies for measuring cytokines

Capability BD CBA ELISA ICS
Allows detection of multiple cytokines in same experiment X   X
Can obtain phenotype of specific cells expressing cytokine of interest     X
Can measure quantity of cytokine secreted X X  

Various Subsets

Intracellular staining of cytokines in cultured Th1, Th2, Th9, Th17, and iTreg cells - 5-color

Detection of three cytokines simultaneously in CD4+ and CD8+ T cells

Cytokine expression in na?ve, memory, and effector T cells (part A)

Cytokine expression in na?ve, memory, and effector T cells (part B)

Intracellular staining of cytokines in cultured Th1, Th2, Th9, Th17, and iTreg cells - 5-color

Transcription factor staining by flow cytometry in two different cell preparations using the BD Pharmingen Transcription Factor Buffer Set

Th17

Intracellular cytokines (human PBMCs)

Th17 culture analyzed using the Human Th1/Th2/Th17 Phenotyping Kit

Intracellular IL-17A and IL-17F staining

Representative data from a time course of Th17-polarized cells comparing levels of IL-17A with CD4, IFN-γ, and IL-4.

Multiplexed quantitation of cytokines in Th17 cultures

RORγt expression on mouse splenocytes cultured under Th17 polarization conditions.

Simultaneous detection of cytokines, transcription factors, and surface markers to characterize mouse Th17 cells

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