Distinguishing Th1 and Th2 Cells
Various techniques differentiate helper T cell subsets
By Jeffrey M. Perkel
Schematic representation of cytokines influencing the development of antigen-activated naive CD4+ T cells into Th1 and Th2 cells
The human body is constantly under siege. It must defend itself from a whole host of bacterial, parasitic, and viral invaders, not to mention rogue cancerous cells. The immune system is the primary line of defense in this game of chess, recognizing and ignoring "self" antigens, while vigorously attacking foreign ones. T lymphocytes, the strategists in this game, both direct the responses of the rest of the immune system and play an active role in it. T cells orchestrate the immune response via the production of cytokines--secreted proteins that instruct other cells to behave in specific ways. Scientists have devoted a great deal of time and effort to understanding the development and differentiation of T lymphocytes because of both the pivotal role T cells play in directing the immune response and the interesting developmental paradigms T cells represent.
T cells may be broadly classified as either helper T cells (Th cells, CD4+) or cytotoxic T cells (Tc cells, CD8+). In 1986, T.R. Mosmann, R.L. Coffman, and colleagues observed that individual clones of helper T cells could be separated into two classes depending upon the specific cytokines the cells secrete in response to antigenic stimulation.1 Th1 cells primarily produce interferon (IFN)-g and interleukin (IL)-2, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13. The two helper T cell classes also differ by the type of immune response they produce. While Th1 cells tend to generate responses against intracellular parasites such as bacteria and viruses, Th2 cells produce immune responses against helminths and other extracellular parasites.2,3 Interestingly, the cytokines produced by each Th subset tend to both stimulate production of that Th subset, and inhibit development of the other Th subset. That is, IFN-g produced by Th1 cells has the dual effect of both stimulating Th1 development, and inhibiting Th2 development. Th2-secreted IL-10 has the opposite effect.4
There is perhaps no more dramatic demonstration of the functional consequence of the diametrically opposing roles of Th1 and Th2 cells than the ability of inbred mouse strains to respond to infection with Leishmania major, an intracellular parasite. Most inbred mouse strains (such as B10.D2) respond to L. major infection with a Th1-like response, clearing the infection. In contrast, BALB/c strains, which respond with a Th2 response, are unable to do so, and ultimately succumb to infection.5
It is unclear as to how newly minted helper T cell precursors can be induced to differentiate into Th1 or Th2 cells in vivo. Cytokines clearly play an important role in the development and/or maintenance of Th lineages. But there is an ongoing debate about exactly what that role is, says Steve Reiner of the University of Pennsylvania. Reiner identifies two competing models: instruction and selection. The instruction model holds that naive T cells are instructed to differentiate into either Th1 or Th2 cells by virtue of the cytokine environment that surrounds them. That is, if naive T cells find themselves in the presence of IL-12, they will differentiate into Th1 cells, whereas IL-4 will stimulate Th2 differentiation. The selection model suggests that the cells randomly "choose" a differentiation path, and the cytokines maintain and select for T cell subsets once they have already committed to a particular fate.
Reiner's lab identifies and distinguishes Th cell subsets using intracellular cytokine staining (ICS). This technique, developed by Andreas Radbruch and colleagues at the University of Cologne, Germany, involves treating cells with Brefeldin A to prevent secretion of intracellular cytokine protein.6 The cells are then permeabilized, tagged with fluorescently labeled antibodies to specific cytokines, and counted using flow cytometry. According to Reiner, the advantage of this technique is that it analyzes the cytokine production of each individual cell as opposed to analyzing the aggregate cytokine production of the entire cell population. Thus, it is possible to see what collection of cytokines each cell is producing. BD Pharmingen of San Diego currently produces an Intracellular Cytokine Staining "starter" kit, containing fixing and permeabilization solutions, as well as labeled antibodies to IL-2, IFN-g, and tumor necrosis factor (TNF)-a. The company also supplies a wide range of other labeled antibodies to supplement this kit, such as antibodies against the various Th2 cytokines. Biosource International of Camarillo, Calif., offers ICScreen? kits for ICS of Th1/Th2 cytokines as well. These kits block cytokine secretion in the Golgi with the Na+ ion-dependent transport inhibitor monensin (ICBlock?). Caltag Laboratories Inc. of Burlingame, Calif., offers the FIX & PERM? cell permeabilization kits and CYTO-IC antibodies designed for ICS.
The major drawback to ICS is that the cells must be killed in order to stain them. Therefore, it is not possible to use ICS to enrich for specific cytokine-producing cell populations for use in downstream applications. In 1995, Radbruch and colleagues described a method to analyze and sort live cells secreting either IgM or IFN-g.7 The authors developed an affinity matrix for the secreted molecule by biotinylating the cell surface, and then binding an antibody-avidin conjugate to it. In a recent publication in Immunity, Kenneth Murphy of the Washington University School of Medicine in St. Louis, and Radbruch (now at Deutsches Rheuma-Forschungszentrum in Berlin, Germany), described a modification of this method, effectively circumventing the shortcomings of ICS.8 The authors replaced the biotin-avidin interaction with a bifunctional antibody conjugate, able to bind to the ubiquitous cell surface marker, CD45, as well as to IL-4. Treatment of the cells with this conjugate created a "high-capacity surface matrix" to bind secreted IL-4. IL-4-secreting cells were identified by the addition of a second anti-IL-4 antibody, conjugated to phycoerythrin (PE). Finally, secreting cells were purified by the addition of anti-PE antibodies conjugated to magnetic microbeads. Miltenyi Biotec Inc. of Auburn, Calif., offers kits based upon this technique, for the enrichment and detection of cells secreting IL-2, IL-4, IL-10, and IFN-g. According to Roger Burger, product manager at Miltenyi Biotec, the IFN-g detection antibody is available in both PE- and FITC-labeled forms, enabling researchers to select for secretion of both IFN-g and IL-10 on the same cell, for example.
Researchers in the lab of Richard M. Locksley at the University of California, San Francisco, use an alternative means of quantifying cytokine-producing cells called the Elispot assay. Cells are cultured in microwell plates coated with an appropriate antibody. As the cells secrete cytokines, these proteins are captured by the nearby plate-bound antibodies. After some period of time, the cells are washed away, alkaline phosphatase-conjugated secondary antibody is added, and the captured cytokines are detected via precipitation of a detection reagent. This produces spots that can be counted, thereby quantifying the number of secreting cells in the assay. Commenting on the differences between ICS and Elispot, Locksley observed that Elispot "tells you the precursor frequency and identifies spontaneously-secreting cells. Intracellular cytokine staining involves restimulating cells, permeabilizing them with saponin and hoping to catch cytokine captured in the ER after poisoning the cells with Brefeldin A. We all do it, but it is relatively insensitive." Elispot detection systems and reagents are offered by R&D Systems of Minneapolis, Biosource International, Mabtech of Nacka, Sweden, and Diaclone of Besan?on Cedex, France. BD Pharmingen, in collaboration with Cellular Technology Ltd. of Cleveland, Ohio, offers both Elispot reagents and high-throughput instrumentation (CTL's ImmunoSpot? Series One Analyzer).
Detecting Transcripts
Though the mechanisms of Th differentiation in vivo are still a mystery, the differentiation of cultured T cells can be readily accomplished in the laboratory. For example, Suneet Agarwal and Anjana Rao, of the Center for Blood Research in Boston, produced the two Th lineages using a pair of mutually exclusive selection conditions.9 Starting with naive (Mel14hi) CD4+ lymphocytes obtained from the spleen and lymph nodes of mice, these authors generated Th1 cells by treating the cells with IL-12 and an antibody against IL-4 (to remove IL-4 from the media). They generated Th2 cells using IL-4, and antibodies against both IFN-g and IL-12. After two weeks, the authors analyzed the cytokine expression profile of each culture using a RiboQuant multiprobe RNAse protection assay (RPA) kit from BD Pharmingen. As expected, after growth under the highly polarizing conditions used in the experiment, the two cultures exhibited a mutually exclusive constellation of cytokines that identified them as either Th1 or Th2. Commenting on the advantages of using RPA to detect transcript levels, Rao says RPA results are "linearly related to the steady-state transcript level, and provide a much more sensitive way of assessing gene transcription [than Northern blots]." Rao adds that, although the RPA kit cannot be used to quantify the level of primary transcript, the technique provides "the next best thing, which is the steady-state level of processed transcript in the cytoplasm."
Th1- and Th2-specific cytokine transcripts may also be detected using RT-PCR. Biosource International offers two Th1/Th2 CytoXpress? multiplex-PCR kits, including amplification reagents (except a thermostable polymerase) and primers. Set 1 simultaneously detects the presence of IFN-g, IL-2, IL-4, and IL-10; set 2 detects each of these, as well as IL-5, IL-12p40, and IL-13. Both sets include GAPDH as a control, and both human and murine versions are available. Biosource also offers PrimeScreen? primer pairs to detect individual cytokines as well.
R&D Systems offers an alternative mRNA detection method with its Quantikine mRNA detection system.10 This system uses two separate oligonucleotide probes: biotin-labeled capture oligonucleotides, and digoxigenin-labeled detection oligonucleotides. RNA samples are hybridized to both probes, and captured to streptavidin-coated microtiter plate wells via the biotin moiety. An anti-digoxigenin alkaline phosphatase conjugate and a colorimetric substrate are used to detect the specific transcripts. According to Leena Martel, a marketing representative at R&D Systems, the Quantikine mRNA kits were originally designed for those customers more comfortable with ELISA kits than with radioactive Northern blots and RPAs. The result is an ELISA-type assay with sensitivity akin to that of a Northern blot, yet which may be performed in less than five hours. One potential drawback of the Quantikine assay is that, for all its sensitivity, it cannot be used to assess either transcript integrity or length, features readily determined by Northern blotting.
Chemicon International's XpressPack? systems function as a cross between Quantikine and RT-PCR techniques. Cytokine gene-specific transcripts are amplified using biotin-labeled PCR primers. The amplified DNA is then denatured and hybridized to a 96-well plate coated with a gene-specific probe. This hybridization is detected by addition of either horseradish peroxidase- or AquaLite?-conjugated streptavidin. XpressPack kits contain the oligonucleotide-coated plates, biotin-conjugated primers, and a positive control; the detection reagents must be purchased separately. Kits are available for the detection of IL-2, IL-4, IL-10, and IFN-g. Tony Endozo, who helped develop the XpressPack kits for Chemicon, notes that the primers included in the kit are designed to be mRNA-specific, so researchers can be certain that amplified products reflect gene expression, and not the presence of genomic DNA. Courtesy of Sigma-Genosys
Nylon membrane-based microarrays present a relatively inexpensive method for multiplexed cytokine transcript detection. These assays provide the benefits of microarrays without the tremendous investment in new technology required for microscope slide-based microarray analysis. In addition, unlike glass arrays, membrane arrays can be stripped and reprobed several times. BD-Pharmingen's RiboScreen? human-1 membrane contains an array of 289 separate human genes on a nylon membrane. Every gene that can be quantified using RiboQuant is represented on the RiboScreen membrane, enabling RiboScreen to be used as an initial screening method prior to RiboQuant analyses. Sigma-Genosys of The Woodlands, Texas and R&D Systems also offer a nylon membrane-based array approach: the Panorama? mouse cytokine gene array contains 514 cDNAs plus controls, while the human cytokine gene array contains 375 genes plus controls. The Common Cytokine-1 GEArrays from SuperArray of Bethesda, Md., contain 23 cytokine genes spotted in duplicate, available for both mouse and human genes. The LifeGrid?, from Incyte Genomics of Palo Alto, Calif., also marketed as the ULTRArray by Ambion of Austin, Texas, features nearly 8,400 human cDNAs. The GeneFilter? from Research Genetics of Huntsville, Ala., contains 5,184 murine or human genes and ESTs. BD Biosciences-CLONTECH of Palo Alto, Calif., offers its popular Atlas? microarrays in a nylon membrane format, with human, mouse, and rat versions, each containing 1,176 genes.
Detecting Proteins
Researchers interested in quantifying the cytokines secreted by a culture have a number of options. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. A comprehensive listing of companies selling antibodies directed against cytokines, which can be used in ELISAs, was previously published in The Scientist.11 Biosource International's ELISA-variant CytoTrap? cell stimulation assay kits can be used to detect secreted Th1/Th2 cytokines. In a CytoTrap assay, cells are grown in an antibody-coated microplate. After stimulation and growth, the cells are washed away, and the secreted cytokines are detected as in a standard ELISA. Unfortunately, purchasing the ELISA reagents necessary for a number of cytokines can be costly. BD Pharmingen offers the human Th1/Th2 cytokine cytometric bead array (CBA) kit, which is a multiplexed flow cytometric assay designed to simultaneously test for IL-2, IL-4, IL-5, IL-10, TNF-a, and IFN-g. The kit uses fluorescent capture beads coated with antibodies against an individual cytokine. The different beads have discrete fluorescent intensities, so they may be distinguished in the FL3 channel of a flow cytometer. Thus, there are six discrete peaks in the FL3 channel, corresponding to each set of capture beads. These beads are mixed with cell culture medium, into which cytokines have been secreted, and then with phycoerythrin (PE)-conjugated secondary antibodies. Their intensity can then be detected in the FL2 channel. According to Deanna Murphy, technical service representative at BD Pharmingen, each bead in the CBA kit acts like an individual ELISA well; the practical implication of this is that each bead in the experiment acts as a replicate "well", so experiments do not need to be set up in duplicate or triplicate.
Courtesy of Upstate Biotechnology
LabMAP? technology from Luminex Corp. of Austin, Texas, also uses beads with differing fluorescent intensities. But rather than using a flow cytometer, results are obtained using Luminex's detection system. The Beadlyte? multi-cytokine detection systems from Upstate Biotechnology of Lake Placid, N.Y.; Bio-Plex? kits from Bio-Rad of Hercules, Calif.; Multiplex Antibody Bead kits from Biosource International; and LINCOplex? cytokine assays from LINCO Research Inc. of St. Charles, Mo., are all based on LabMAP technology. Courtesy of Upstate Biotechnology
Beadlyte cytokine detection systems are sandwich immunoassays performed on unique fluorescent beads, which contain a precise distribution of two fluorescent dyes, and a specific cytokine capture antibody linked to its surface. The fluorescent bead sets are incubated with a biological sample. A biotinylated cytokine reporter antibody is then used to identify the captured cytokine bound to the specific fluorescent bead. The reaction is detected by adding a streptavidin-phycoerythrin conjugate to the reaction mixture and read in a Luminex 100 instrument.
BioErgonomics Inc. of St. Paul, Minn., offers MultiFlow? kits capable of detecting up to three different cytokines at once, using paramagnetic beads. Unlike other systems, these beads are not fluorescent; the analytes are identified using fluorescently tagged antibodies instead. Because the light-scattering properties of these beads are distinct from cells, the beads can be distinguished from cells during flow cytometry. This allows researchers to mix beads with the cells and analyze both at the same time. Multiplexed assays will be discussed in greater detail in the May 28, 2001, issue of The Scientist.
Helper T cells play a central role in directing the immune response. Whether an antigen elicits a Th1- or Th2-type response has a profound effect on the ability of the body to clear the infection. Scientists will therefore continue to use the tools discussed in this article to study helper T cells as models for cellular development and differentiation.
Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com.
References
1. T.R. Mosmann, et al., "Two types of murine helper T cell clone: I. Definition according to profiles of lymphokine activities and secreted proteins," Journal of Immunology, 136:2348-57, 1986.
2. A. O'Garra, N. Arai, "The molecular basis of T helper 1 and T helper 2 cell differentiation," Trends in Cell Biology, 10:542-50, Dec. 2000.
3. T.R. Mosmann, R.L. Coffman, "Th1 and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties," Annual Review of Immunology, 7:145-73, 1989.
4. A.K. Abbas, et al., "Functional diversity of helper T lymphocytes," Nature, 383:787-93, 1996.
5. S.L. Reiner, R.M. Locksley, "The regulation of immunity to Leishmania major," Annual Review of Immunology, 13:151-77, 1995.
6. M. Assenmacher, et al., "Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin-10 in interferon-g and in interleukin-4-expressing cells." European Journal of Immunology, 24:1097-101, 1994.
7. R. Manz, et al., "Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix," Proceedings of the National Academy of Science, 92:1921-5, 1995.
8. W. Ouyang, et al., "Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment," Immunity, 12:27-37, 2000.
9. S. Agarwal, A. Rao, "Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation," Immunity, 9:765-75, 1998.
10. H.E. Sussman, "A quantum leap in mRNA quantitation," The Scientist, 14[21]:22, Oct. 30, 2000.
11. B. Sinclair, "The divine cytokine," The Scientist, 14[7]:36, April 3, 2000.
