Dealing from the Evolutionary Pawnshop
Authors: Steven L Reiner, Robert A Seder
Journal: Immunity (1999)

Cell fate decisions are made throughout the embryogenesis of higher vertebrates in order to pattern a complex multicellular organism. In the adult organism, cell fate decisions are continually made by lymphocytes to ensure homeostasis in the uninfected individual and during defense against pathogenic microorganisms. Helper T cells make numerous decisions during ontogeny, postdevelopmental circulation in lymphoid organs, and immune response. The decision concerning which type of effector cell to become upon antigenic challenge has been reexamined closely in the past year. The results of these findings will serve as a model of what has become an increasingly exciting area of complex, developmentally regulated gene expression. It is hoped that this speculative and hypothetical portrait of lymphocytes will help focus further discussion and investigation about the evolution of cellular decision making in higher species. To put the current problem in perspective, it would be useful to review the history of the decisions that were required to supply us with an uncommitted, naive helper T cell. First, an evolutionary decision was made 450 million years ago. This was the selective survival of individuals harboring the RAG genes. It is now generally accepted that the two closely linked genes, which encode the central lymphocyte-specific enzymatic activities necessary for V(D)J recombination, are probably derived from a retrotransposase (3Agrawal A Eastman Q.M Schatz D.G Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system.Nature. 1998; 394: 744-751Crossref PubMed Scopus (586) Google Scholar, 33Hiom K Melek M Gellert M DNA transposition by the RAG1 and RAG2 proteins a possible source of oncogenic translocations.Cell. 1998; 94: 463-470Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). The irony, of course, is that the transposable element containing the RAG genes, virtually stolen from microbes, is essential for existence in a microbial world and completely nonessential for normal development, growth, and reproduction in a clean environment, isolated from most microbes. The definitive T cell lineage arises from multipotent hematopoietic stem cells. Thymic development includes the first of several major differentiation steps made by T cells. This first step is the decision that results in specificity. Through the process of V(D)J recombination, unique antigen receptors are expressed on individual T cells. This process is stochastic although perhaps not completely random. V(D)J recombination is also associated with the decision to become an αβ versus γδ T cell. The next differentiative step occurs when double-positive αβ thymocytes select between the CD4+ and CD8+ single-positive fates. This decision is exclusive in the sense that cells have exited the cell cycle (24Ernst B Surh C.D Sprent J Thymic selection and cell division.J. Exp Med. 1995; 182: 961-971Crossref PubMed Scopus (78) Google Scholar) and, therefore, one double-positive cell can adopt only one of the two fates (Figure 1). The best-characterized cell fate decision of the peripheral naive CD4+ helper T cell appears to be a choice of the Th1 or Th2 fate. Several recent developments in this field now prompt us to reexamine the current models of how this decision is made. In the periphery, a naive helper T cell that becomes activated generally undergoes rapid cellular proliferation. In the context of cell division, each clonal T cell population begins making decisions about functional (effector) differentiation. It is now evident that IL-2, an autocrine growth factor that has powerful proliferative effects on activated T cells, is expressed without entry into the cell cycle (8Bird J.J Brown D.R Mullen A.C Moskowitz N.H Mahowald M.A Sider J.R Gajewski T.F Wang C.-R Reiner S.L Helper T cell differentiation is controlled by the cell cycle.Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). By contrast, the cytokines that characterize effector lineages are regulated by the cell cycle. The first expression of interferon-γ (IFNγ), the signature gene of Th1 cells, requires entry into the S phase of the cell cycle. IL-4, the signature gene of Th2 cells, requires greater cell division (at least three cycles) to be expressed (8Bird J.J Brown D.R Mullen A.C Moskowitz N.H Mahowald M.A Sider J.R Gajewski T.F Wang C.-R Reiner S.L Helper T cell differentiation is controlled by the cell cycle.Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). For some cytokines, even greater than four cell divisions may be required for their expression (28Gett A.V Hodgkin P.D Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation.Proc. Natl. Acad. Sci. USA. 1998; 95: 9488-9493Crossref PubMed Scopus (280) Google Scholar). It should be noted that effector differentiation in B cells (isotype switching) is also regulated by cell division (37Hodgkin P.D Lee J.H Lyons A.B B cell differentiation and isotype switching is related to division cycle number.J. Exp. 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There has been some link established between V(D)J recombination and the cell cycle (79Schlissel M Constantinescu A Morrow T Baxter M Peng A Double-strand signal sequence breaks in V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell cycle regulated.Genes Dev. 1993; 1: 2520-2532Crossref Scopus (334) Google Scholar, 55Li Z Dordai D.I Lee J Desiderio S A conserved degradation signal regulates RAG-2 accumulation during cell division and links V(D)J recombination to the cell cycle.Immunity. 1996; 5: 575-589Abstract Full Text PDF PubMed Scopus (167) Google Scholar). It is unclear whether cell division is required to generate combinatorial diversity or whether it is simply due to the large arrays of possible gene segments. For diversity to be generated in the effector repertoire of a clonal B cell or T cell, however, there must be cell division in order to yield progeny bearing the same receptor but different maturational fates (Figure 1). The differentiation decision of helper T cells is highly regulated by cytokines (66O'Garra A Cytokines induce the development of functionally heterogeneous T helper cell subsets.Immunity. 1998; 8: 275-283Abstract Full Text Full Text PDF PubMed Scopus (1319) Google Scholar). Despite the dramatic ability of excesses in IL-12 and IL-4 signaling in generating polarized Th1- and Th2-predominant populations, respectively, the cytokine interventions have very little effect on generating large percentages of Th1- and Th2-committed cells among the earliest cell divisions following T cell activation (8Bird J.J Brown D.R Mullen A.C Moskowitz N.H Mahowald M.A Sider J.R Gajewski T.F Wang C.-R Reiner S.L Helper T cell differentiation is controlled by the cell cycle.Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Visualization of this phenomenon has been greatly facilitated by techniques that allow for the single-cell analysis of cytokine expression. One of the most striking but underemphasized observations in the initial differentiation process is the remarkable inefficiency of acquiring an effector phenotype (13Bucy R.P Karr L Huang G.Q Li J Carter D Honjo K Lemons J.A Murphy K.M Weaver C.T Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.Proc. Natl. Acad. Sci. USA. 1995; 92: 7565-7569Crossref PubMed Scopus (121) Google Scholar, 8Bird J.J Brown D.R Mullen A.C Moskowitz N.H Mahowald M.A Sider J.R Gajewski T.F Wang C.-R Reiner S.L Helper T cell differentiation is controlled by the cell cycle.Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Thus, it is probably the case that in a population of cells uniformly signaled to assume a given fate (TCR signaling and either recombinant IL-12 or IL-4), this inefficiency reflects a rate-limiting step in the initial activation of effector cytokine genes. The rate-limiting nature of effector cytokine expression is similar to the phenomenon known as position-effect variegation (89Wakimoto B.T Beyond the nucleosome epigenetic aspects of position-effect variegation in Drosophila.Cell. 1998; 93: 321-324Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), owing to an inaccessible state of the chromatin. In Drosophila eye development, a fly can develop a uniformly and completely colored eye (i.e., all cells are the same color) if the gene responsible for the eye color is in an accessible chromatin environment. In some cases, however, the eye will develop into one that has only a small number of eye cells with that color, the rest being a different color. Importantly, the variegated state can arise without mutating the primary sequence of the color gene or any gene product necessary to transactivate the gene, including signaling and DNA-binding proteins specific for the color gene. The variegated effect can result merely from transposition of the eye color gene proximate to a less-accessible chromatin environment. Position-effect can be engineered in lymphocytes when positive cis-acting sequences are deleted from a uniformly expressed gene, resulting in an infrequent or variegated pattern of expression (25Festenstein R Tolaini M Corbella P Mamalaki C Parrington J Fox M Miliou A Jones M Kioussis D Locus control region function and heterochromatin-induced position effect variegation.Science. 1996; 271: 1123-1125Crossref PubMed Scopus (306) Google Scholar). The initial cell divisions following polarized T cell differentiation appear variegated. All cells may be signaled to become either Th1 or Th2 cells, but only small numbers initially adopt effector fates (13Bucy R.P Karr L Huang G.Q Li J Carter D Honjo K Lemons J.A Murphy K.M Weaver C.T Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.Proc. Natl. Acad. Sci. USA. 1995; 92: 7565-7569Crossref PubMed Scopus (121) Google Scholar, 8Bird J.J Brown D.R Mullen A.C Moskowitz N.H Mahowald M.A Sider J.R Gajewski T.F Wang C.-R Reiner S.L Helper T cell differentiation is controlled by the cell cycle.Immunity. 1998; 9: 229-237Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). A model predicting that Th1 and Th2 cells arise from a common precursor cell expressing multiple cytokines (45Kamogawa Y Minasi L.A Carding S.R Bottomly K Flavell R.A The relationship of IL-4- and IFNγ-producing T cells studied by lineage ablation of IL-4-producing cells.Cell. 1993; 75: 985-995Abstract Full Text PDF PubMed Scopus (266) Google Scholar) is seemingly at odds with a model suggesting variegated initial appearances of effector lineages. This might reflect insensitivity of single-cell gene expression assays. Alternatively, the multipotential precursor may contain incomplete or sterile cytokine transcripts from inaccesible genes or may promiscuously activate cytokine promoters because reporter transgenes are isolated from negative cis-acting elements of the endogenous gene. The initial variegation or inefficiency of helper T cell commitment that rapidly, over subsequent cell divisions, gives way to polarized populations raises the question of whether cytokines act in a selective or an instructive manner. In other words, does the differentiative cytokine actually produce a signal that alters the gene expression pattern of a cell (instruction) or does it merely enrich a population by favoring the growth or death of subsets that arise in a cell-autonomous, stochastic manner (selection) (Figure 2)? Importantly, both models argue that cytokines play a prominent (or dominant) role in the outcome of effector development. Single-cell analyses have demonstrated that even highly polarized populations are often contaminated with cells of the opposing fate (48Kelso A Groves P Troutt A.B Francis K Evidence for the stochastic acquisition of cytokine profile by CD4+ T cells activated in a T helper type 2-like response in vivo.Eur. J. Immunol. 1995; 25: 1168-1175Crossref PubMed Scopus (81) Google Scholar) that may not have been apparent when secreted cytokines were measured from culture supernatants. The inability to fully homogenize a population may reflect an experimental or biological inefficiency in the instructive process, but it may also be an indicator that helper T cells have a significant degree of autonomy in making decisions. The recent findings that double-deficient STAT-4/STAT-6 mutant mice can generate Th1 cells (46Kaplan M.H Wurster A.L Grusby M.J A signal transducer and activator of transcription (Stat)4-independent pathway for the development of T helper type 1 cells.J. Exp. Med. 1998; 188: 1191-1196Crossref PubMed Scopus (136) Google Scholar) argues against an essential role for IL-12 in instruction of the Th1 lineage. In addition, the findings that naive human T cells can be primed in the absence of IL-4 to become Th2-like (20Demeure C.E Yang L.P Byun D.G Ishihara H Vezzio N Delespesse G Human naive CD4 T cells produce interleukin-4 at priming and acquire a Th2 phenotype upon repetitive stimulations in neutral conditions.Eur. J. Immunol. 1995; 25: 2722-2725Crossref PubMed Scopus (92) Google Scholar, 44Kalinski P Hilkens C.M Wierenga E.A van der Pouw-Kraan T.C van Lier R.A Bos J.D Kapsenberg M.L Snijdewint F.G Functional maturation of human naive T helper cells in the absence of accessory cells. Generation of IL-4-producing T helper cells does not require exogenous IL-4.J. 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These results parallel the differentiation of erythroid cells, where lineage commitment clearly occurs without instruction from erythropoietin signaling and instead selection (survival and proliferation) of the lineage is impaired (94Wu H Liu X Jaenisch R Lodish H.F Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (832) Google Scholar, 56Lin C.S Lim S.K D'Agati V Costantini F Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.Genes Dev. 1996; 10: 154-164Crossref PubMed Scopus (341) Google Scholar). While some data support a selective role for cytokines in influencing helper T cell differentiation, at least three questions remain unresolved. The first is whether IFNγ- and IL-4-expressing cells that develop in the absence of STAT-4 and STAT-6, respectively, constitute the same differentiative fate as bona fide Th1 and Th2 cells. A second issue is whether cytokines may act in an instructive manner above and beyond a selective role (Figure 2). This possibility is raised by analogy to B cell differentiation in which cytokines such as IL-4 clearly play some role in the commitment of cells to specific isotype switching (53Lebman D.A Coffman R.L Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures.J. Exp. Med. 1988; 168: 853-862Crossref PubMed Scopus (272) Google Scholar, 16Coffman R.L Lebman D.A Rothman P Mechanism and regulation of immunoglobulin isotype switching.Adv. Immunol. 1993; 54: 229-270Crossref PubMed Scopus (460) Google Scholar). 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The observed association of chromatin remodeling in a population of cells conditioned by cytokines could mean either the cytokine causes cells to remodel a locus or the cytokine merely promotes expansion of cells with a remodeled locus (2Agarwal S Rao A Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation.Immunity. 1998; 9: 765-775Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Based on studies of the initial infrequency in commitment (13Bucy R.P Karr L Huang G.Q Li J Carter D Honjo K Lemons J.A Murphy K.M Weaver C.T Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.Proc. Natl. Acad. Sci. 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It is possible that enrichment of a particular subset may involve negative effects on an opposing subset. Such a negative effect might be instructive or selective. For example, IL-4 may enrich for Th2 cells by impairing the growth of already committed Th1 cells (negative selection) or by inhibiting the number of progeny that can commit to the Th1 lineage (negative instruction) (Figure 2). The possibility that helper T cell differentiation is initially stochastic also prompts us to speculate whether there is any true default to the helper T cell response in the absence of factors such as IL-12 and IL-4. If the process is intrinsically diversified for fates, then the finding that Th1 cells predominate after in vitro culture of stimulated STAT-4/STAT-6 double-knockout cells (46Kaplan M.H Wurster A.L Grusby M.J A signal transducer and activator of transcription (Stat)4-independent pathway for the development of T helper type 1 cells.J. Exp. 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