T Helper. Antigen-presenting cell

Introduction CD4 T cells play a central role in immune protection. They do so through their capacity to help B cells make antibodies, to induce macrophages to develop enhanced microbicidal activity, to recruit neutrophils, eosinophils, and basophils to sites of infection and in? ammation, and, through their production of cytokines and chemokines, to orchestrate the full panoply of immune responses.

Beginning with the groundbreaking work of Mossman and Coffman in 19861 showing that long-term CD4 T-cell lines could be subdivided into 2 groups, those that made IFN as their signature cytokine and those that produced IL-4, it has been realized that CD4 T cells are not a unitary set of cells but represent a series of distinct cell populations with different functions. While some of these CD4 T-cell populations are actually distinct lineages of cells already distinguished from one another when they emerge from the thymus, such as “natural” regulatory T (nTreg) cells2,3 and

natural killer T cells (NKT cells),4 several represent alternative patterns of differentiation of naive CD4 T cells. It is to the description of these cells, their functions, their patterns of differentiation, the sets of genes they express, and the consequences of abnormalities in them that this review is devoted. Naive conventional CD4 T cells have open to them 4 (and possibly more) distinct fates that are determined by the pattern of signals they receive during their initial interaction with antigen. These 4 populations are Th1, Th2, Th17, and induced regulatory T (iTreg) cells.

Mossman and Coffman recognized the Th1 and Th2 phenotypes among the set of long-term T-cell lines that they studied and the early history of this ? eld was devoted to understanding these 2 cell populations, with Th1 cells being regarded as critical for immunity to intracellular microorganisms and Th2 cells for immunity to many extracellular pathogens, including helminths. 5,6 Abnormal activation of Th1 cells was seen as the critical event in most organ-speci? c autoimmune diseases while Th2 cells were responsible for allergic in? ammatory diseases and asthma.

Th17 cells have been recognized much more recently but there is now a growing body of work indicating not only that these cells exist but that they play a critical function in protection against microbial challenges, particularly extracellular bacteria and fungi. 7 Further, some of the autoimmune responses formally attributed to Th1 cells, such as experimental autoimmune encephalomyelitis (EAE), collagen induced arthritis (CIA), and some forms of in? ammatory bowel disease (IBD), have now been shown to be mediated, at least in part, by Th17 cells.

iTreg cells are also now well established as an inducible cell population that phenotypically resembles nTreg cells, although distinguishing the function of iTreg cells from that of nTreg cells and, particularly, the relative importance of the 2 Treg populations in humans and experimental animals has been dif? cult. In this review, we will deal with the function of Treg cells as a group except where we explicitly speak of iTreg cells. There are also other regulatory CD4 T cells including Th3 and TR1 cells.

Th3 cells are transforming growth factor (TGF- )–producing cells induced by oral tolerance. 8 Most of them are likely inducible regulatory T cells that express Foxp3. 9 Whether or not there are TGF -producing Foxp3 CD4 T cells is unclear. TR1 cells are IL-10 producing cells. 10 Because all the CD4 T-cell sets including Th1, Th2, Th17 as well as Treg cells are capable of producing IL-10 under certain circumstances,11-13 TR1 cells may not be a distinct lineage but rather may represent a certain state of each existing lineage.

Finally, there may well be other sets of conventional CD4 T cells and even among the more conventional sets, important differences exist, such as the detailed pattern of cytokines that they produce. Figure 1 summarizes much of what we know about the major sets of CD4 T cells, including their unique products, the characteristic transcription factors and cytokines critical for their fate determination and some of their functions. Each of these topics will be discussed in some depth in the subsequent sections of this review. A little history

Initially, immunologists believed that there were fundamentally 2 types of immune responses that require the action of CD4 T cells. One was antibody-mediated and the other cell-mediated. However, there was very little progress in this area until the early 1980s, when T-cell cloning technology was developed, many cytokines were discovered and cloned, and assays for them became available. Submitted May 6, 2008; accepted June 9, 2008; DOI 10. 1182/blood-200805-078154. BLOOD, 1 SEPTEMBER 2008 VOLUME 112, NUMBER 5 1557 1558 From bloodjournal.

hematologylibrary. org at NARODNY ONKOLOGICKY USTAV on May 22, 2012. For personal use only. ZHU and PAUL BLOOD, 1 SEPTEMBER 2008 VOLUME 112, NUMBER 5 Figure 1. Summary of the 4 CD4 T helper cell fates: their functions, their unique products, their characteristic transcription factors, and cytokines critical for their fate determination. Tim Mosmman and Bob Coffman recognized that mature CD4 T cells could be subdivided into 2 distinct populations with different sets of products and that this would endow them with unique functions.

1 Kim Bottomly was also working on this subject; she and her colleagues subdivided CD4 T-cell lines based on functional criteria, distinguishing in? ammatory and helper CD4 T cells, with the latter being IL-4 producers. 14 The translation of the differences observed in long-term CD4 T-cell lines to the behavior of normal CD4 T cells, ? rst in vitro and then in vivo, constitutes the beginning of the Th ? eld as a biologic subject. The earliest description of in vitro differentiation was reported in 1990 by our group and that of Susan Swain, demonstrating ?

rst that naive CD4 T cells failed to make IL-4 (or most other effector cytokines) and that these cells could be induced to develop into vigorous IL-4 producers if they were stimulated both with T-cell receptor ligands and IL-4, itself. 15,16 Within 2 to 3 days after the initiation of culture, the stimulated cells acquire the capacity to produce IL-4. It was subsequently shown that this in vitro differentiation requires a signaling pathway that includes the IL-4 receptor, the signal transducer and activator of transcription (Stat) 6 and the DNA-binding factor GATA-3.

17,18 As we will discuss later, this is far from the whole story, but “it gets us off to the races. ” We note in passing that in our original 1990 paper, we found that IL-2 was also necessary for cells to acquire IL-4–producing capacity, although that was largely overlooked and didn’t come back for serious analysis for more than a decade. 19 Three years later, Ken Murphy, Anne O’Garra, and their colleagues showed that naive CD4 T cells could acquire the capacity to produce IFN in vitro.

20 They stimulated T-cell receptor transgenic naive CD4 T cells and antigen-presenting cells with cognate antigen and heat-killed Listeria monocytogenes organisms; the heat-killed Listeria caused cells in the culture to produce IL-12, which was critical for Th1 differentiation in this system. At ? rst, it appeared that there was a fundamental dichotomy between the logic of differentiation process for Th1 and Th2 cells, with a CD4 T-cell endogenous product, IL-4, playing a major positive feedback role in Th2 differentiation and an exogenous product, IL-12, probably mainly from dendritic cells, playing the major inductive role for Th1 cells.

However, with time and attention, the logic of the differentiation processes appears to be much closer than initially appreciated. Neutralizing IFN strikingly diminishes Th1 differentiation; IL-12 appears to induce some IFN production which then acts to up-regulate the key transcription factor T-bet21,22 and leads to much more IFN production, showing a positive feedback loop for Th1 cells as well. Immunologists attributed many autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and their experimental models, to the action of Th1 cells.

However, they were puzzled by the paradoxical ? nding that neutralizing or knocking out IL-12 and IFN had different effects on the induction of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. IL-12 p40 knockout mice are resistant to EAE induction whereas IFN knockout mice are more sensitive. The discovery of IL-23, which consisted of IL-12p40 paired with a distinctive chain (p19), led to a reassessment of the relative contributions of IL-12 and IL-23 in EAE induction. 23 Indeed, it is IL-23, not IL-12, that plays the major role in inducing EAE.

Due to the linkage between IL-23 and the expression of IL-17, a new Th lineage, Th17, was soon identi? ed. 24,25 Th17 cells are different from classical Th1/Th2 cells based on the following evidence: Th17 cells do not produce the “classical” Th1/Th2 cytokines; Th17 cells express low levels of T-bet and GATA-3; and the Th1/Th2 signature cytokines, IL-4 and IFN , suppress Th17 cell differentiation. 24,25 In 2006, Stockinger, Weaver, Kuchroo, and their colleagues each showed that Th17 cells could be induced in vitro from naive mouse CD4 T cells by stimulation through their T-cell receptor (TCR) in the presence of IL-6 and TGF- .

26-28 ROR t was identi? ed as the master regulator gene for Th17 cells. 29 More work has revealed that the role of TGF- in human cells may not be central to Th17 differentiation but that IL-1 has an important role. 30,31 However, very recently, 3 groups independently reported that TGF- was also critical for human Th17 cell differentiation. 32-34 The discrepancy between these reports and previous studies may be explained by the potentially different purity of the naive T-cell population each group prepared because a small contamination with effector/memory cells may suppress de novo Th17 cell differentiation.

In addition, in the earlier studies, the amount of TGF- added to the culture and/or present in the serum is much higher than the amount required for Th17 differentiation and high levels of TGF- inhibit Th17 cell differentiation and favor iTreg differentiation. IL-21 produced by Th17 cells, induced in the course of Th17 differentiation,35-37 ful? lls the role of the powerful positive feedback stimulant, reinforcing the Th17 induction process and showing that Th17 development has the logic similar to that of Th1 and Th2 cells. The Treg “revolution” has been one of the de?

ning themes of modern immunology but reaching an understanding of how these cells differentiate has been complex. In 1995, Sakaguchi and his colleagues discovered that regulatory T cells express CD25. 38 Transfer of CD4 T cells that had been depleted of the CD25 population into congenitally athymic mice induced autoimmune diseases while transfer of intact populations of CD4 T cells did not. In 2001, the autoimmune Scurfy mice and a human immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) patient were found to have mutations in Foxp3.

39-41 In 2003, Foxp3 was reported as the master transcriptional regulator for nTreg cells. 42,43 Weiner and colleagues had reported in 1994 that oral tolerance regimens induced TGF- –producing CD4 T regulatory cells. 8 This cell population was designated Th3 cells. In 2003, Chen et al reported that TGF- can convert Foxp3 naive CD4 T cells into Foxp3 CD4 T cells, that is iTreg cells. 44 It is now clear that activated naive CD4 T cells stimulated by TGF- in the absence of proin? ammatory cytokines develop into iTreg cells. The positive feedback factor here is TGF- itself, although there is still much

BLOOD, 1 SEPTEMBER 2008 VOLUME 112, NUMBER 5 From bloodjournal. hematologylibrary. org at NARODNY ONKOLOGICKY USTAV on May 22, 2012. For personal use only. CD4 T CELLS: FATES, FUNCTIONS AND FAULTS 1559 uncertainty as to the relative biologic importance of nTreg and iTreg cells, particularly in humans. Converting the Th paradigm from in vitro to in vivo situations initially met with much resistance but with time it became clear that memory and memory/effector T cells from normal priming events do display polarization in their cytokine-producing capacity, in their functions and in the range of cell surface molecules they express.

Indeed, the recent description of the selective de? cit in development of Th17 cells in patients with hyper-IgE syndrome (HIES or Job syndrome) strikingly validates this concept. 45 HIES patients have a genetically determined inability to signal through Stat3, due to dominant negative mutations in the SH2 domain or the DNA-binding domain of this molecule. 45-47 In humans and mice, the 3 major inducers and/or sustainers of Th17 differentiation, IL-6, IL-21 and IL-23, each use Stat3 for signal transduction. Indeed, the principal dif?

culties HIES patients face, recurrent staphylococcal and fungal infections, are precisely those observed in mice that cannot develop Th17 cells, strikingly validating the importance of the CD4 T-cell differentiation concept and indicating that lessons are learned, although not always perfectly, by studying experimental animals. Th cells: cytokine produced and functions Th cells play critical roles in orchestrating the adaptive immune responses. They exert such functions mainly through secreting cytokines and chemokines that activate and/or recruit target cells.

Th1 cells mediate immune responses against intracellular pathogens. 5,6 In humans, they play a particularly important role in resistance to mycobacterial infections. Th1 cells are also responsible for the induction of some autoimmune diseases. Their principal cytokine products are IFN , lymphotoxin (LT ), and IL-2. IFN produced by Th1 cells is important in activating macrophages to increase their microbicidal activity. 48 LT has been implicated as a marker for the disease progression in multiple sclerosis patients. 49 LT -de? cient mice are resistant to EAE.

50 IL-2 production is important for CD4 T-cell memory. IFN IL-2 cells are regarded as precursors of the Th1 memory cells. 51 IL-2 stimulation of CD8 cells during their priming phase is critical for CD8 memory formation. 52 Th2 cells mediate host defense against extracellular parasites including helminths. 5,6 They are important in the induction and persistence of asthma and other allergic diseases. Th2 cells produce IL-4, IL-5, IL-9, IL-10, IL-13, IL-25, and amphiregulin. IL-4 is the positive feedback cytokine for Th2 cell differentiation15,16 and is the major mediator of IgE class switching in B cells.

53 IgE binds to Fc RI on basophils and mast cells and, when interacting with a multivalent ligand, cross-links Fc RI, leading to the secretion of active mediators such as histamine and serotonin and to the production of several cytokines including IL-4, IL-13, and tumor necrosis factor (TNF- ). IL-5 plays a critical role in recruiting eosinophils. 54 In addition to its effect on mast cells and lymphocytes, IL-9 induces mucin production in epithelial cells during allergic reactions. 55 IL-10, produced by Th2 cells, suppresses Th1 cell proliferation. 56 IL-10 can also suppress dendritic cell function.

57 IL-13 is the effector cytokine in the expulsion of helminths and in the induction of airway hypersensitivity. 58,59 Amphiregulin is a member of the epidermal growth factor (EGF) family. It induces epithelial cell proliferation. In the absence of amphiregulin, the expulsion of the nematode Trichuris muris is delayed. 60 Amphiregulin may also be important for the induction of airway hypersensitivity. IL-25 (also known as IL-17E) is also a Th2 cytokine. 61,62 IL-25, signaling through IL-17RB, enhances the production of IL-4, IL-5, and IL-13 by a unique c-kit Fc RI nonlymphocyte population.

63 Interestingly, IL-25 is also produced by lung epithelial cells in response to allergens. 55 Thus, IL-25 serves as an initiation factor as well as an ampli? cation factor for Th2 responses. IL-25 can induce the production of chemokines including RANTES (CCL5) and eotaxin (CCL11) that recruit eosinophils. Th17 cells mediate immune responses against extracellular bacteria and fungi. 7 They are responsible for, or participate in, the induction of many organ-speci? c autoimmune diseases. Th17 cells produce IL-17a, IL-17f, IL-21, and IL-22.

IL-17a was originally cloned as CTLA-8 and is homologous to a Herpesvirus saimiri gene. It was renamed IL-17 when its receptor was cloned. 64 IL-17a and IL-17f are genetically linked and presumably under the control of the same locus control region (LCR). Thus, IL-17a and IL-17f are often coexpressed at the single cell level although there are also IL-17a- and IL-17f-single producing cells, suggesting the regulation of IL-17a and IL-17f expression in Th17 cells mirrors that of IL-4 and IL-13 in Th2 cells (see below).

IL-17a and IL-17f both use the IL-17RA chain for their signaling, implying that they have similar functions, although IL-17a binds to IL-17RA with much higher af? nity. 65 IL-17a can induce many in? ammatory cytokines, IL-6 as well as chemokines such as IL-8 (also known as CXCL8), and thus has an important role in inducing in? ammatory responses. 64 Both IL-17a and IL-17f recruit and activate neutrophils during immune responses against extracellular bacteria and fungi. IL-21 made by Th17 cells is a stimulatory factor for Th17 differentiation and serves as the positive feedback ampli?

er,35-37 as does IFN for Th1 and IL-4 for Th2 cells. IL-21 also acts on CD8 T cells, B cells, natural killer (NK) cells, and dendritic cells. 66 IL-22 is produced by Th17 cells through IL-6– or IL-23–mediated Stat3 activation67; TGF- inhibits IL-22 expression. 13 The aryl hydrocarbon receptor (AHR), a receptor for dioxin, is highly expressed in Th17 cells and plays an important role in the expression of IL-22. 68 IL-22 mediates IL-23–induced acanthosis and dermal in? ammation. 67 IL-22 also protects hepatocytes during acute liver in? ammation.

69 Strikingly, IL-22 mediates host defense against bacterial pathogens such as Klebsiella pneumoniae70 and Citrobacter rodentium. 71 However, these functions may largely depend upon IL-23 stimulation of innate cells to produce IL-22 rather than on the action of Th17 cells. 71 Treg cells play a critical role in maintaining self-tolerance as well as in regulating immune responses. 2 Increasing Treg numbers and/or enhancing their suppressive function may be bene? cial for treating autoimmune diseases and for preventing allograft rejection.

Indeed, Treg cells stimulated in vitro with alloantigen prevent both acute and chronic allograft rejection in mice. 72 On the other hand, depletion of Treg cells and/or inhibition of their function could enhance immunity against tumors and chronic infectious agents. Treg cells exert their suppressive functions through several mechanisms, some of which require cell-cell contact. 3 The molecular basis of suppression in some cases is through their production of cytokines, including TGF- , IL-10, and IL-35. TGF- produced by Treg cells may also result in the induction of iTreg cells from naive CD4 T cells.

Although TGF- is not absolutely required for suppression in some settings, particularly in vitro, it is very important in mediating suppression in several circumstances in vivo. 73,74 IL-10 production is critical for Treg-mediated prevention and cure of in? ammatory bowel disease. 75,76 Speci? c deletion of 1560 From bloodjournal. hematologylibrary. org at NARODNY ONKOLOGICKY USTAV on May 22, 2012. For personal use only. ZHU and PAUL BLOOD, 1 SEPTEMBER 2008 VOLUME 112, NUMBER 5 IL-10 in Treg cells by Foxp3-Cre results in the development of spontaneous colitis and enhanced lung in? ammation.

77 IL-10 also plays an important role in limiting the severity of EAE at later stages. During Leishmania infection, Treg IL-10 production in the lesion maintains a homeostasis between the host and the pathogen, allowing a low level of pathogen persistence and a consequent continued stimulation of protective immunity. 78 IL-35, which consists of EBI3, a chain shared with IL-27, and IL-12 p35, is produced by Treg cells and contributes to suppressive activity. 79 CD4 T cells other than Th2 and Treg cells can also produce IL-10. IL-10 production by Th1 or Th17 cells may play an important role in limiting their own effector function.

11-13 IL-10, IL-27, and TGF can induce IL-10 production. 10,13,80 Interestingly, Foxp3-deleted “Treg cells,” judged by expression of GFP encoded by a Foxp3null locus, produce high levels of IL-10, suggesting that IL-10 production in Treg cells is independent of Foxp3. 81 The originally described TR1 cells (IL-10–producing regulatory T cells) may include many different types of cells that are capable of producing IL-10. Thus, IL-10 production by all CD4 T cells serves as a negative regulatory mechanism for limiting the immune responses to prevent host tissue damage.

Th17 cells. Surprisingly, there has been little study of the expression of TGF R on various Th cells. Among the chemokine receptors, human Th17 cells coexpress CCR6 and CCR4. 97 Treg cells The majority of the nTreg cells express CD25. 2 Although all activated T cells express CD25, Treg cells express the highest levels of CD25 and do so constitutively, whereas expression by conventional CD4 T cells is transient and lower. The high level of expression of CD25, IL-2R , on Treg cells suggests the importance of IL-2 for these cells. Treg cells also express CTLA-4, GITR, and Folr4.

However, these markers are only useful for distinguishing Treg cells from naive conventional CD4 T cells because each can be induced by activation of conventional T cells. Treg cells, especially in human, express little or no IL-7R . The absence of IL-7R in combination with high levels of CD25 provides an approach to identifying Treg cells and separating them from other cells. 98 An interesting subset of Treg cells, those that express CD103,99 also known as alpha E integrin, is mainly found in the gut or at sites of in? ammation. Most iTreg cells induced in vitro express CD103. Expression of cytokine and chemokine receptors by Th cells

Th1 cells Transcription factors critical for each T helper lineage Transcription factors including members of the nuclear factor of activated T cell (NFAT), NF- B, and activator protein-1 (AP-1) families are critically involved in cytokine production upon TCR and/or cytokine stimulation. Presumably, those factors are also important during the process of T helper differentiation. However, they are not the factors directly determining T helper lineage fates and are usually expressed in all lineages. Below, we will focus on the transcription factors that either are speci? cally expressed, or function differently, in each of the lineages.

Transcription factors for Th1 differentiation IL-12R 2 expression is induced by TCR activation and then maintained by IL-12 as well as by IFN stimulation. 82-84 IL-12R 1 is constitutively expressed on naive CD4 T cells and its expression is further increased in Th1 cells through an IRF1-dependent mechanism. 85 Up-regulation of the IL-12R complex conveys IL-12 hyperresponsiveness to activated cells. IL-18R is also upregulated during Th1 differentiation. Although IL-18 is not involved in the differentiation of Th1 cells, it can synergize with IL-12 in inducing IFN , implying that IL-18 plays an important role in Th1 responses.

86,87 Although chemokine receptor expression and differentiated Th phenotype are not strictly coordinate, some receptors, such as CXCR388,89 and CCR5,90 show a striking preferential expression on Th1 cells. Th2 cells IL-4R is up-regulated by IL-4 during Th2 differentiation. However, other c cytokines may also induce IL-4R . CD25 (IL-2R ) expression is higher in Th2 cells than in Th1 cells, possibly due to the action of c-Maf. 91 Such higher expression of CD25 may confer hyperresponsiveness to IL-2.

The most important cell surface marker for Th2 cells is T1/ST2 (IL-33R ).

92 T1/ST2, also known as IL-1R like 1, belongs to the IL-1R superfamily, which includes IL-1R and IL-18R . The function of IL-33R on Th2 cells may mirror the function of IL-18R on Th1 cells. Among the chemokine receptors, CCR3,93 CCR4,88,89 CCR8,94 and CRTh295 tend to be expressed on Th2 cells. Th17 cells Th17 cells express high levels of IL-23R. 27,31,37 In addition, Th17 cells express substantial amounts of IL-1R1 and of IL-18R . The function of IL-18R on Th17 cells is unclear while IL-1R1 appears critical for IL-17 production; mice de? cient in IL-1R1 are resistant to EAE, which is correlated with reduced IL-17 production.

96 This is also consistent with a requirement for IL-1 in induction of human T-bet,21 the Th1 master regulator, is up-regulated during Th1 differentiation. Stat1, the major transducer of IFN signaling, plays a critical role in the IFN -mediated induction of T-bet. 22 Overexpression of T-bet in Th2 cells induces them to produce IFN and inhibits their production of IL-4. T-bet / cells have severe defects in Th1 cell differentiation. T-bet / mice spontaneously develop asthma-like diseases. 100 However, T-bet / Th1 cells still produce some IFN .

Eomesodermin (Eomes),101 another T-box family member critical for IFN production in CD8 T cells, is up-regulated during Th1 differentiation, suggesting that it may also be involved in IFN production by CD4 T cells. Indeed, IL-21 treatment of Th1 cells partially inhibits IFN production, correlating with suppression of Eomes but not T-bet. 102 Stat4, an IL-12 signal transducer, is important for amplifying Th1 responses. 103,104 In addition, Stat4 can directly induce IFN production in activated CD4 T cells, which can initiate the positive feedback loop in which IFN , acting through T-bet, induces more IFN .

IL-12/Stat4, together with an NF- B inducer, can cause IFN production independent of TCR stimulation. This is best illustrated by the capacity of IL-12 and IL-18, whose receptor is expressed on Th1, but not Th2, cells to induce IFN production by Th1 cells in a cyclosporine A–independent matter. 86,87 BLOOD, 1 SEPTEMBER 2008 VOLUME 112, NUMBER 5 From bloodjournal. hematologylibrary. org at NARODNY ONKOLOGICKY USTAV on May 22, 2012. For personal use only. CD4 T CELLS: FATES, FUNCTIONS AND FAULTS 1561 Runx3,105,106 a transcriptional repressor important for silencing CD4 during CD8 T-cell development, is also up-regulated in Th1 cells.

Overexpression of Runx3 in Th2 cells induces IFN production independent of T-bet (our unpublished data). Runx3-de? cient cells produce less IFN than wild type Th1 cells. 106 Hlx, a transcription factor induced by T-bet, interacts with T-bet and enhances T-bet-mediated IFN production. 107 Transcription factors for Th2 differentiation Stat6, activated by IL-4, is the major signal transducer in IL-4– mediated Th2 differentiation. 108-110 Stat6-de? cient cells fail to develop IL-4–producing capacity in vitro; in vivo, Th2 responses independent of Stat6 activation can be obtained. 111-113 In vitro, Stat6 activation is necessary and suf?

cient for inducing high expression levels of the Th2 master regulator gene, GATA-3. 114,115 Overexpression of GATA-3 in Th1 cells induces IL-4 production116 and in the absence of GATA-3, Th2 differentiation is totally abolished in vitro and in vivo. 117,118 Even in fully differentiated Th2 cells, deleting GATA-3 completely blocks the subsequent production of IL-5 and IL-13,117 although it has only a modest effect on IL-4 production, consistent with the presence of GATA-3-binding sites in the promoters of IL-5 and IL-13 but not in the IL-4 promoter. There are 2 Stat5 family members, Stat5a and Stat5b.

119 They are important for cytokine-driven cell proliferation and cell survival. IL-2 potently stimulates Stat5 activation. Th2 cell differentiation requires strong Stat5 signaling. 19,120 Thus, Stat5a single knockout cells have profound defects in Th2 cell differentiation both in vitro and in vivo despite the presence and activation of Stat5b. Stat5 has been shown to directly bind to DNase I hypersensitive sites (HSII and HSIII) in the second intron of the Il4 locus. 120 c-Maf, which is selectively up-regulated in Th2 cells, also enhances IL-4 production but does not play a role in the production of other Th2 cytokines.

121 IRF-4 expression is required for Th2 cell differentiation. 122,123 IRF-4–de? cient cells produce much less IL-4, but this defect can be rescued by overexpression of GATA-3, suggesting that IRF-4 up-regulates GATA-3. 122 G? -1 is an immediate early IL-4–inducible gene. 124 TCR activation also transiently induces G? -1 expression. G? -1 selects GATA-3hi cells for growth by modulating both the upstream and the downstream IL-2 signaling events. 124,125 Transcription factors for Th17 differentiation Figure 2. T-cell differentiation involves instructive differentiation as well as selective expansion of differentiated cells.

The cytokines critical for the differentiation of each lineage instruct activated CD4 T cells to express their master transcription factors, T-bet for Th1, GATA-3 for Th2 and ROR t for Th17, as well as other lineage speci? c factors, IL-12R for Th1, G? -1 for Th2 and IL-23R for Th17. In many instances, only a portion of cells expresses the indicated transcription factors and adopts the differentiated phenotype. Such differentiated cells express the factors that determine responsiveness to particular cytokines, IL-12 for Th1, IL-2 for Th2 and IL-23 for Th17 cells, thus leading to selective expansion of those differentiated cells.

Transcription factors for Treg differentiation As noted above, most patients with IPEX and Scurfy mice have FOXP3/Foxp3 mutations, which result in loss of functional Treg cells. Overexpression of Foxp3 in conventional T cells converts them to a Treg phenotype and endows them with anergy and suppressive activity. 42 TGF- induces Foxp3 expression. 44 Continuous expression of Foxp3 is critical for maintaining the suppressive activity of Treg cells. 131 Diminishing the degree of Foxp3 expression may convert Treg cells to Th2 like cells, implying a close relationship of the Th2 and Treg lineages.

132 Stat5 activation by IL-2, important for Th2 differentiation, is also required for Treg development. 133 Stat5 may contribute to Foxp3 induction through binding to its promoter. 134,135 T helper differentiation Th1 cell differentiation ROR t is important in Th17 cell differentiation. 29 Overexpressing ROR t induces IL-17 production, whereas ROR t-de? cient cells produce very little IL-17. Indeed, ROR t-de? cient mice are partially resistant to EAE. Another related nuclear receptor, ROR , is also up-regulated in Th17 cells. 126 Although ROR dele