Instruction, Selection, or Tampering with the Odds?

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Science  21 May 1999:
Vol. 284, Issue 5418, pp. 1283-1285
DOI: 10.1126/science.284.5418.1283

Lymphocytes are a late evolutionary addition to the immune system of vertebrates, enabling effective host defense against a wide variety of pathogenic microbes. The effector cells of the immune system [HN1], T and B lymphocytes [HN2], undergo two distinct modes of differentiation; one endows them with specificity for a particular antigen, the other with specific effector functions (TH1 versus TH2 cytokine patterns for CD4+ T cells; specific antibody classes for B cells). Recombination of the V, D, and J segments of antigen receptor genes [HN3] results in the generation of populations of T and B lymphocytes that express a broad repertoire of antigen receptors. In this way, the immune system is able to respond to a wide array of pathogens. Binding of antigen to the corresponding receptor stimulates the lymphocyte bearing that receptor to differentiate. In the case of T cells, functional differentiation is accompanied by distinct expression patterns for genes encoding cytokines [HN4] and surface receptors. When stimulated by antigen, precursor T cells bearing the CD4 [HN5] marker [T helper (TH) cells] differentiate into either TH1 cells or TH2 cells [HN6]. TH1 cells produce interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β) [HN7] and protect against intracellular pathogens; TH2 cells, which produce interleukin (IL)-4, IL-5, and IL-13 [HN8], help to control extracellular pathogens, and mediate allergy [HN9] (1).

The best characterized influence on the differentiation of TH cells is the cytokine environment [HN10] (1). TH cells first activated by antigen in the presence of IL-12 [HN11] develop predominantly into TH1 cells, whereas those activated in the presence of IL-4 develop predominantly into TH2 cells. There is a debate about whether the cytokines that induce TH1 or TH2 differentiation “instruct” the developmental fate of naïve TH cells or “select” for cells that through a random (stochastic) process of gene activation already produce a combination of cytokines indicative of a TH1 or TH2 cell (see the figure).

Modeling T helper cell differentiation.

In the instructive model of TH cell differentiation, all daughter cells adopt one of two developmental states specified by the presence or absence of specific differentiative signals (upper panel). In the selection model, daughter cells adopt differentiation states in a random (stochastic) manner (middle panel). Pure populations can subsequently arise if one cell type is selectively expanded or maintained in preference to the other. In a model that combines both instruction and selection, the ratio of the two types of daughter cells varies depending on exposure to extrinsic differentiation inducing cytokines (lower panel). If the shift in the ratio of the two cell types is extreme, pure populations could be generated without further selection. Pink and purple circles represent two alternate differentiation states.

Several observations published in the past year offer new, somewhat surprising, insights into the way in which differentiative inducer cytokines regulate TH cell differentiation. Using fluorescent tags to record the number of cell divisions of individual cells, naïve TH cells were shown to require a specified number of cell divisions before becoming competent to produce cytokines indicative of either the TH1 or TH2 pathway. Different numbers of divisions are required for different cytokines (2, 3). Furthermore, stable cytokine expression is accompanied by demethylation and increased chromatin accessibility of the cytokine genes (methylation [HN12] is an epigenetic mechanism for silencing genes) (2, 4). The most unexpected finding is that IL-2, IL-4, and, possibly, other cytokines are expressed from only one of two alleles in many individual TH cells [HN13] (5). These seemingly unrelated findings do not clearly resolve the simple question of instructed differentiation versus random differentiation and selection; instead they suggest that elements of both models may contribute to TH cell differentiation.

Cell proliferation appears necessary for the differentiation of TH subsets. The initial expression of both TH1 and TH2 cytokines is cell cycle-dependent, but IFN-γ expression appears during the initial cell division whereas IL-4 requires at least three divisions (2). Furthermore, lineage commitment in the earliest stages of differentiation is strikingly inefficient and heterogeneous. Even under optimal conditions for either TH1 or TH2 differentiation, the number of cells that express subset-specific cytokine genes is low and is invariably accompanied by low frequencies of cells expressing atypical cytokine patterns. Thus, the TH cell fate decision has intrinsic heterogeneity, and sibling cells can develop different, even opposite, phenotypes.

Gene loci for effector cytokines such as IL-4, IL-13, and IFN-γ are epigenetically repressed in naïve T cells (their chromatin [HN14] structure is closed and their cytosines methylated) (2, 4). The link between transcriptional derepression and the cell cycle [HN15] most likely reflects the opportunity for synthesis of new DNA that is less methylated and more accessible than that of the parent strand (6). After differentiation, epigenetic changes and allelic expression patterns persist for multiple cell divisions, showing that they are both stable and inheritable. Thus, TH1 and TH2 lymphocytes exhibit memory of both specificity and function, reflecting the stability of both types of differentiation. The mechanisms of this memory, however, are different. Stability of clonal specificity results from genetic recombination, whereas stability of function is accomplished by epigenetic modification.

Why should cytokine genes be expressed from only one allele? The clearest examples of monoallelic expression of autosomal genes are found in two organ systems for which cellular specificity and population diversity are essential: the immune system (antigen receptors on T, B, and natural killer cells) and the nervous system (olfactory receptors on olfactory epithelial cells) (7). Monoallelic expression (or allelic exclusion) ensures that most individual cells express only one member of a family of receptors encoded by highly homologous genes, resulting in each cell having only one of many possible specificities. The functional significance of monoallelic expression of cytokine genes is less obvious and may simply reflect the rate limitations of chromatin remodeling at these loci.

The question of instructive versus selective differentiation has been addressed in other hematopoietic [HN16] cell lineages. Both instruction and selection have been reported for various lineage-restricted growth and differentiation factors (8). For example, macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor [HN17] appear to instruct different fates in bipotential granulocyte-macrophage progenitors. In contrast, erythropoietin [HN18] supports expansion of cells committed to the erythroid lineage (selection) but is not required for progenitor cells to make that commitment. Perhaps the example that is most relevant to TH differentiation is the analogous functional differentiation of B cells involving isotype switch recombination of immunoglobulin heavy chain genes [HN19]. Cytokines (including IL-4 and IFN-γ) can dramatically alter the switching between immunoglobulin isotypes in differentiating B cells (9). Antibody isotype switching requires transcriptional activation of heavy chain genes, and IL-4 and IFN-γ regulate switching by inducing transcription of specific heavy chain genes. The result of cytokine action on differentiation is, thus, instructive, not selective. This instruction takes the form of changes in the probability of different outcomes within the intrinsic constraints of a stochastic process. For example, the probability that B cells will switch to IgE ranges from <0.0001 to >0.01 in the absence or presence, respectively, of IL-4.

The regulation of TH1 and TH2 differentiation by differentiative inducer cytokines such as IL-12 or IL-4 may not be adequately described as either strictly instructive or strictly selective. The rate-limiting nature of chromatin remodeling of cytokine gene loci introduces an element of probability into the process, much as it does for antibody isotype switching for B cells. Cytokine inducers of TH1 or TH2 differentiation could alter the odds of stable chromatin remodeling of specific cytokine gene loci. Extremes of TH1 or TH2 differentiation may be achieved either by subsequent selection or by large changes in probability (see the figure). To decide among these models, it may prove necessary to produce the equivalent of an embryologist's fate map, accounting for the birth, differentiation, and death of all descendants of an individual T cell stimulated under highly controlled conditions.

HyperNotes Related Resources on the World Wide Web

General Hypernotes

Cell & Molecular Biology Online, maintained by P. Gannon, is a well-organized collection of annotated links to Internet resources.

The On-line Medical Dictionary is offered by CancerWEB.

HealthWeb maintains a collection of links to immunology resources on the Web.

The New Zealand Rheumatology Association (NZRA) provides a glossary of terms used in immunological research.

The Immunology Book Case, provided by the Dalhousie University Medical School, provides information on immunological topics.

The Biology Project of the University of Arizona presents an introduction to immunology.

S. Baron, University of Texas Medical Branch at Galveston, presents an Immunology Overview by A. Goldman and B. Prabhakar, which is the first section of the Web textbook Medical Microbiology.

J. Brown, Section of Microbiology, Department of Molecular Biosciences, University of Kansas, provides lecture notes for an immunology course. Images of immune system molecules are provided.

C. Hewitt, Department of Microbiology and Immunology, University of Leicester, UK, offers an illustrated introduction to immunology in lecture notes for a course on infection and immunity. A Glossary of Immunology is provided by the Department's Infection & Immunity Web page.

N. Holmes, Immunology Division of the Department of Pathology, University of Cambridge, UK, provides lecture notes for an immunology course.

Kimball's Biology Pages offers essays by J. Kimball on immunology and other topics in molecular and cellular biology.

Specific Immunity is a chapter of the Microbiology 101/102 Internet Text provided by R. Hurlbert, Department of Microbiology, Washington State University, Pullman, for an introductory course in microbiology.

COPE (Cytokines Online Pathfinder Encyclopaedia) is an extensive hypertext resource on cytokines and related topics presented by H. Ibelgaufts, Institute of Biochemistry, Ludwig-Maximilians-University of Munich. (Note: The links to specific COPE entries in the Numbered Hypernotes may be valid for a limited time only because entry page URLs change with each new version of the resource.)

The Howard Hughes Medical Institute presents an immunology overview and articles about the immunology research of HHMI investigators.

Numbered Hypernotes

1. The Virtual Explorer from the Wilson Group at the University of California, San Diego, offers an illustrated introduction to the cells and the molecules and proteins of the immune system. The On-Line Biology Book from Estrella Mountain Community College, Avondale, AZ, includes a unit on the lymphatic system and immunity and links to Web resources on immunology. R. Falk, Section of Plant Biology, University of California, Davis, provides an overview of the immune system in lecture notes for a biology course. The Fajerlab Web page at Florida State University provides an introduction to the immune defense system for a biology course. As part of a lecture series on immunology, the Natural Toxins Research Initiative, Texas A&M University, Kingsville, presents an introduction to the cells and tissue of the immune response.

2. T cell and B cell are defined in the On-line Medical Dictionary. The Immunology Book Case includes presentations on T cells and B cells. N. Holmes provides lecture notes about B cells and T cells for an immunology course. Kimball's Biology Pages offers a presentation on T and B cells.

3. V genes, D genes, and J genes are defined in the NZRA immunology glossary. S. Arkins, Department of Biological Sciences, Illinois State University, discusses VDJ gene segments in lecture notes titled “Generation of antibody diversity: Molecular mechanisms” (continued in the next lecture) for an immunology course. Kimball's Biology Pages includes an essay on antigen receptor diversity.

4. The On-line Medical Dictionary defines cytokines. COPE provides an introduction to cytokines and includes a list of cytokine topics as entry points for topical browsing in the encyclopedia. The Immunology Book Case presents an introduction to cytokines. A brief introduction to cytokines is provided by the Chapter Resources pages for Kuby's Immunology. The Cytokines Web provides scientific information about cytokines and their receptors, including 3D structural information and topological and evolutionary relationships. J. Brown provides lecture notes on cytokines for an immunology course.

5. The On-line Medical Dictionary defines CD4. COPE has entries for CD antigens and CD4.

6. The NZRA immunology glossary defines helper (TH) cells. T helper cell, TH1 cells, and TH2 cells are defined in the On-line Medical Dictionary. Kimball's Biology Pages offers an introduction to T helper cells. The Immunology Book Case provides information on T helper cells and their subsets. C. Hewitt discusses T helper cells in lecture notes titled “Immunology: Cell and cytokine networks.” COPE provides an overview of TH1/TH2 cytokines.

7. COPE includes articles about interferons and tumor necrosis factor and entries for IFN-gamma and TNF-beta. The THCME Medical Biochemistry Page has sections on interferon-gamma and tumor necrosis factor-beta.

8. The On-line Medical Dictionary has entries for interleukin-4, interleukin-5, and interleukin-12. COPE provides an introduction to interleukins and entries for IL-4, IL-5, and IL-13.

9. In his chapter on specific immunity, R. Hurlbert discusses the role of T helper cells in developing immunity. C. Hewitt provides illustrated lecture notes on the effector mechanisms of the immune response. The MEdIC (the Medical Education Information Center) Web site offers a presentation on the role of cytokines and T helper cells in allergic diseases. A tutorial by M. Browning on the role that CD4-positive T cells play in bacterial killing is offered by the Infection and Immunity Homepage of the Department of Microbiology and Immunology, University of Leicester, UK.

10. A presentation by E. Palmer, Committee on Immunology, University of Chicago, titled “T cell immune responses” is available from the Science Partners for Teachers Web site. J. Brown discusses helper T cell differentiation in lecture notes on T cell subsets, superantigens, and cytokines. N. Holmes discusses the differentiation of T helper cells in lecture notes titled “Cell interactions: Cytokines” for an immunology course. An article by M. Haak-Frendscho and J. Balwit titled “Revolutions in the TH1/TH2 paradigm of T helper cell subsets: Implications for the future” appeared in Promega Notes Magazine, Number 45, 1994. The 19 July 1996 issue of Focus, a newsletter published by Harvard Medical School, had an article by G. Strobel about the research of M. Grusby and L. Glimcher on the molecular basis of the differentiation of T helper cells.

11. The On-line Medical Dictionary defines interleukin-12. The Immunology Book Case has a section on interleukin-12. COPE has an entry for IL-12.

12. The On-line Medical Dictionary defines DNA methylation. P. McClean, Department of Plant Sciences, North Dakota State University, Fargo, discusses methylation in lecture notes on the control of gene expression in eukaryotes for a course on intermediate genetics. G. Podgorski, Department of Biology, Utah State University, Logan, discusses DNA methylation in lecture notes on the regulation of gene expression for a course on developmental biology.

13. The On-line Medical Dictionary defines interleukin-2. The Immunology Book Case has an entry for interleukin-2. COPE has an entry for interleukin-2. The 27 March 1998 issue of Science had a Research Commentary by A. Chess titled “Expansion of the allelic exclusion principle?” about the research of G. Holländer and colleagues on the monoallelic expression of the mouse gene encoding IL-2.

14. Chromatin is defined in the On-line Medical Dictionary. The Chromatin Structure & Function Page is provided by J. Bone, University of Texas M. D. Anderson Cancer Center. The Cell Biology Graduate Program, University of Texas Medical Branch, offers a presentation by G. Childs on the organization of nuclear chromatin. R. Stewart, Dosimetry Research and Technology Group, Pacific Northwest National Laboratory, Richland, WA, makes available an essay titled “A few words about DNA and chromatin.” For a lecture and laboratory course on molecular biology, G. Lindquester, Department of Biology, Rhodes College, Memphis, TN, provides lecture notes on chromatin and methylation.

15. Cell Division and the Cell Cycle, made available by the Department of Biological Sciences, University of Alberta, describes the cell cycle and includes a glossary of terms. COPE has an article on the cell cycle.

16. COPE includes an article about hematopoiesis. J. Brown presents lecture notes on the hematopoietic system.

17. The On-line Medical Dictionary defines macrophage-colony stimulating factor and granulocyte-macrophage colony stimulating factor. COPE provides an overview of colony stimulating factors and has entries for macrophage colony stimulating factor and granulocyte colony stimulating factor.

18. The On-line Medical Dictionary defines erythropoietin and erythroid progenitor cells.

19. The NZRA immunology glossary defines isotype. S. Arkins, Department of Biological Sciences, Illinois State University, provides lecture notes on isotype switching for an immunology course. COPE includes an article on isotype switching.

20. R. L. Coffman is in the Department of Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA, which is a division of the Schering-Plough Research Institute.

21. S. L. Reiner is in the Gwen Knapp Center for Lupus and Immunology Research, University of Chicago.


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