- Email: [email protected]
T helper cell effector fates — who, how and where?
T helper cell effector fates — who, how and where? R Lee Reinhardt, Suk-Jo Kang, Hong-Erh Liang and Richard M Locksley CD4 helper T cells functionally organize the host immune response by elaborating cytokines, often in patterns that have overlapping effects on other cells. Much interest centers on understanding how these stereotyped cytokine patterns become elaborated and what mechanisms underlie the generation of distinct helper T cell subsets. The past two years have seen advances in understanding of additional subsets, including T helper follicular cells and IL-17-producing T helper cells. Progress has also been achieved in resolving some of the crosstalk that regulates effector fate at the level of distinct transcription factors and chromatin reorganization of the cytokine genes, and a crucial role for gene silencing has been exposed. Finally, the role of innate cells in influencing these processes has become increasingly realized. Addresses University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0795, USA Corresponding author: Locksley, Richard M ([email protected])
Current Opinion in Immunology 2006, 18:271–277 This review comes from a themed issue on Lymphocyte activation Edited by Bernard Malissen and Janet Stavnezer Available online 17th April 2006 0952-7915/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2006.03.003
Introduction CD4 helper T (Th) cells orchestrate host immunity by secreting cytokines; these act on immune and nonimmune cells to regulate the response to pathogens. Naı¨ve CD4 T cells are maintained in a pluripotent state and have a relatively quiescent effector program as they recirculate from blood through lymphoid organs surveying dendritic cells for activating MHC–peptide complexes. Through complex mechanisms that integrate signals from activated dendritic cells and from the cytokine milieu, naı¨ve T cells are driven through rapid rounds of division that are linked intimately with the acquisition of the capacity to secrete effector cytokines necessary to confront distinct groups of pathogens. These events have been correlated with epigenetic changes in DNA and chromatin surrounding the cytokine genes, which reflects their competent or silenced states [1,2]. With time, daughter cells become fully differentiated and fixed in www.sciencedirect.com
their effector lineages and migrate to sites where their cytokines functionally organize the immune response. In this review, we will focus on recent studies that have identified new subsets of Th cells, new insights regarding the activation and/or silencing of discrete cytokine networks in these subsets, and investigations into the role of dendritic cells and other innate cells in regulating the differentiation, trafficking and/or maintenance of Th subsets.
The who — new Th subsets Additional Th subsets have been characterized that have effector functions distinct from the canonical Th1 (interferon [IFN]g-secreting) and Th2 (interleukin [IL]-4secreting) subsets; these include Tr1 (IL-10-secreting), Th3 (transforming growth factor [TGF]b-producing), ThFH (follicular helper cells), peripherally-induced T regulatory (Treg; FoxP3-positive) and Th17 (IL17A-producing) cells (Figure 1). Although the increasing amount of literature on Treg will not be covered in this review, new information concerning ThFH and Th17 cells has increased our understanding of these subsets and will be discussed. ThFH cells
ThFH cells, which were first identified in humans, represent T cells found in germinal centers that possess a cytokine profile distinct from Th1 and Th2 cells. ThFH cells have a different transcriptional profile compared with Th1 and Th2 cells, and selectively express CD84, CD200, B cell lymphoma (Bcl-6) and IL-2 [3,4]. Although subsets of ThFH cells have been described, we define ThFH cells to be cells that express CXCR5 and that migrate to B cell follicles after activation. T cell–B cell interactions at the follicular border are crucial for efficient B cell activation and for antibody production, and migration to the border is dependent upon the expression of chemokine receptors by both T and B cells. CXCR5 is responsible for ThFH cell migration and a recent study suggests that a push–pull mechanism positions these cells [5]. In CXCR5-deficient mice, antigen-specific T cells were retained in the lymph node T cell areas by CCR7; this suggests that competition between two chemokine receptors positions activated T cells. Imaging studies support the evidence that activated B cells modulate chemokine receptor expression and move to the follicular border in a CCR7-dependent manner, where they engage T cells and actively pull them into the Current Opinion in Immunology 2006, 18:271–277
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Figure 1
T helper subsets. Although the precise lineage of the subsets in blue has not been defined by cell fate-mapping experiments, Th1, Th17 and Th2 cells represent alternative effector cell fates individually derived from naı¨ve precursor T cells (Thp). The degree to which these cell fates are conditioned by different classes of dendritic cells (DC) is unknown. The differentiation, maintenance and effector functions of these subsets share certain symmetries. Th1 cells express T-bet and IL-12 receptors (IL-12R) downstream of IFNg signals, and are further maintained by IL-12 and IL-18, members of the greater IL-6-like and IL-1-like superfamilies, respectively. Th17 cells develop in the presence of TGFb and IL-6, and are further maintained by IL-23 and IL-1b, members of the same superfamilies. Th2 cells are maintained by IL-4 itself, as well as by the IL-1 superfamily member IL-33. Downstream, Th subsets recruit distinct innate cell types to mediate effector functions in the periphery.
follicle [6]. Another study, which used isolated human ThFH cells and activated B cells, indicated that ThFHmediated B cell help could come via the B cell itself [7]. This study suggests that B cells can modulate the expression of certain costimulatory and chemokine receptors on ThFH cells, but this depends on the activation status of the B cell and on the presence of antigen. Indeed, recent literature supports the idea that multiple costimulatory molecules and their ligands on T and B cells play important roles in optimal antibody production and in class switching. Inducible costimulatory molecule (ICOS) is one such molecule. ICOS-deficient mice or mice treated with an antibody against its receptor had a reduced number of ThFH cells [8]. CXCR5 expression on CD4 T cells was linked to ICOS expression and was enhanced by interactions with B cells. CD4 T cells from mice that have a mutated roquin gene expressed high levels of ICOS and these animals had elevated numbers of ThFH cells [9]. These mice also had abnormally high numbers of germinal centers and eventually developed autoimmune disease, which highlights the potential importance of ThFH cells in regulating autoantibody production. As both IL-4-secreting and IFNg-secreting Th cells have been identified in follicles, the precise lineage of ThFH cells as a unique or terminally differentiated state of Th subset development remains unclear. Current Opinion in Immunology 2006, 18:271–277
Th17 cells
Th17 cells have been linked to model autoimmune diseases including experimental allergic encephalomyelitis and collagen-induced arthritis [10,11]. Th17 cells secrete IL-17A, IL-17F, IL-6 and tumor necrosis factor (TNF)a, and their functions reflect the capacities of these cytokines collectively to mobilize, to recruit and to activate neutrophils, in part by induction of chemokines and growth factors from diverse cell types. Two recent studies [12,13] support a hypothesis wherein these cells arise as a separate Th lineage, and predict that they do not represent a population of Th1 cells that have undergone further differentiation. When naı¨ve CD4 T cells were cultured in the presence of antiIFNg during priming, a large percentage of the cells generated were Th17 cells. An additive effect was observed when IL-4 was blocked, which implies that the development of Th17 cells is inhibited by the presence of IFNg and IL-4. Further evidence that these are a unique lineage of Th cells came from experiments that showed that cells from mice deficient in T-box expressed in T cells (T-bet), signal transducer and activator of transcription 4 (Stat4) or Stat6 had no defect in IL-17 production. Thus, traditional regulators of Th1 and Th2 cells did not appear to be www.sciencedirect.com
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necessary for Th17 differentiation. The transcription factors T-bet and GATA-binding protein 3 (GATA3), which have been associated with Th1 and Th2 differentiation, respectively, were not required for Th17 development in vivo [14]. Interestingly, this study showed that TGFb produced by CD4 Treg cells in the presence of IL-6 was responsible for Th17 differentiation, and that the further addition of IL-1b or TNFa led to highly efficient differentiation of these cells. Although not required for their development, IL-23 was shown to be important in the survival and maintenance of Th17 cells. Lund et al. [15] found Th1 and Th2 mRNA expression profiles were altered when naı¨ve cells were cultured in the presence of TGFb. It will be interesting to see if any of the 20 genes that they determined to be regulated by TGFb might be a transcription factor that acts as a ‘master regulator’ for the Th17 lineage.
The how – signal integration, feed-forward amplification and crosstalk Signal strenth
Activation of alternative cytokine fates during naı¨ve CD4 T cell priming has been attributed to alterations in the ‘strength of signal’ layered onto extensive cross-regulation between the lineages. Accumulating evidence has implicated the nuclear transcription factor (NF)-kB and extracellular signal-related kinase (ERK) cascades in modulating signal strength. Schnurri-2 is a transcriptional repressor that competes with p50 for binding to consensus NF-kB motifs. Schnurri-2-deficient T cells demonstrated constitutive NF-kB activation and enhanced Th2 differentiation, which correlated with increased induction of GATA3 [16]. Conversely, T cells deficient in the NF-kB family member Bcl-3 had decreased GATA3 induction and Th2 differentiation, perhaps reflecting the ability of the Bcl-3–p50 complex to bind and transactivate the GATA3 gene in in vitro assays [17]. It will be interesting to see whether Schnurri-2 represses the GATA3 promoter by antagonizing Bcl-3–p50 complexes in Th2 cells. Cells from mice deficient in the NF-kB family member RelB demonstrated impaired Th1 differentiation, which correlated with deficiencies in T-bet and Stat4 induction after activation. Transient ERK activation by weak TCR–CD28 signals induced IL-2R-dependent Stat5 phosphorylation, GATA3 expression and IL-4 production; these were abolished by stimulation with high antigen doses that induced sustained ERK activation [18]. Pharmacologic ERK inhibitors decreased GATA3 protein levels in developing Th2 cells by enhanced ubiquitin–proteosome degradation. ERK activation stabilized GATA3 protein levels, and Mdm2 could act as an E3 ligase for GATA3 [19]. Further work is needed to assess whether ERKmitogen activated protein kinase (MAPK)-mediated phosphorylation of GATA3 is responsible for these effects. ERK signals also mediate GATA3-dependent www.sciencedirect.com
histone H3 acetylation of Th2 cytokine loci, suggesting that this pathway influences GATA3 function at multiple levels. T-bet versus GATA3
One observation in the T-bet knockout mouse was the tendency of animals to spontaneously develop Th2mediated inflammation. During Th1 priming, T-bet was phosphorylated on Tyr525 by the Tec kinase Itk. Although Tyr525 phosphorylation of T-bet did not affect activation of IFNg, this modification facilitated a physical interaction of T-bet with GATA3, resulting in its sequestration from binding sites in the Th2 cytokine locus [20]. At low levels of TCR–CD28 activation, however, Itk was required for efficient GATA3 induction and for Th2 development [21], indicating that further complexities underlie the crosstalk between these master regulators. An additional modification of T-bet involved Ser508 phosphorylation by casein kinase I and glycogen synthase kinase-3. This modification led to attenuation of IL-2 production as it promoted heterodimers of T-bet and RelA, thus interfering with RelA activation of the IL-2 promoter [22]. Additional targets of T-bet continued to be identified. Osteopontin is a T-bet-dependent gene in Th1 cells [23], and the transcription factor Hlx is a Tbet-induced gene that interacts physically with T-bet in a feed-forward mechanism required for optimal activation, but not maintenance, of the IFNg locus [24]. Conditional GATA3 knockout cells were used to demonstrate the non-redundant role for GATA3 in both initiating and maintaining Th2 cell fate [25,26]. GATA3 function could be attenuated by PU.1, an ETS family transcription factor implicated in myeloid and B cell fate decisions, which was induced transiently in a Stat6dependent manner after Th2 priming [27]. Intriguingly, PU.1 induction was variegated in the population and interfered with GATA3 DNA binding; it is possible that this contributed to allelic diversity in the spectrum of expressed Th2-family cytokines [28]. Sequence mining
Novel regulatory elements continue to be revealed by mining sequences across the Th1 and Th2 cytokine loci. A major finding was the identification of a rad50embedded element upstream of IL-13 and IL-4; this functioned as a locus control region that had both enhancer and chromatin-modifying activity in mediating Th2 cytokine expression by long-range intrachromosomal interactions [29,30]. This locus control region was demonstrated to undergo indiscriminate modification after T cell priming, although an additional DNase I hypersensitivity site developed in response to Stat6-mediated signals [31]. Dynamic mapping of chromatin alterations across the Th2 locus indicated that DNase hypersensitivity site IV, which is 30 of Il4, is a repressive element [32]; this was confirmed by targeted deletion [33]. Th1 Current Opinion in Immunology 2006, 18:271–277
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cells from silencer-deficient mice continued to produce IL-4, and were unable to use efficient Th1-mediated immunity to clear pathogens such as Leishmania major. Silencing in the Th2 locus of Th1 cells was associated with methylated H3 accumulation mediated by the Polycomb family methyltransferase EZH2 [34]. Intriguingly, intergenic transcription could be detected in the silenced Th2 locus, raising the possibility that RNA-based silencing mechanisms might occur [35]. Initial evaluation of Dicer-deficient CD4 cells revealed inappropriate IFNg production during Th differentiation [36]. Progress has also been made in identifying elements that regulate activation of the Ifng gene. Enhancer elements 5 kb upstream and 18 kb downstream of the gene underwent histone modifications during Th1 priming [37], and the upstream element was demonstrated to bind NF-AT and T-bet in chromatin immunoprecipitations [38]. T-bet was demonstrated to bind directly at the Ifng promoter utilizing conserved T-box half-sites to transactivate the gene in cooperation with CCAAT/ enhancer binding protein b [39,40]. Enforced T-bet expression could dissociate the mSin3a co-repressor from the endogenous locus, consistent with the capacity of Tbet to transactivate the Ifng gene when introduced into Th2 cells. A striking finding was an interchromosomal association between regulatory elements of the Th2 cytokine locus on chromosome 11 and the Ifng promoter on chromosome 10 in the mouse. Th differentiation was correlated with chromosomal reorganization that favored intrachromosomal interactions, suggesting that this physically underpins alternative cytokine expression patterns [41]. It would be interesting to consider these interactions in natural killer T cells — an atypical lineage that co-expresses IFNg and IL-4.
The where — instruction or selection? Interest continues to surround the issue of how differentiation of Th substrates is regulated: is this controlled by dendritic cells conditioned in response to distinct pathogens or is there selection by a more stochastically regulated process by which primed T cells respond to cytokines in the environment, which promote their subsequent survival and growth. Activation of a TLR11dependent pathway in dendritic cells by a Toxoplasma profilin-like molecule led to IL-12 induction, which potentially accounts for the strong Th1 response to infection by this organism [42]. Conversely, MyD88-deficient mice developed aberrant Th2 responses to L. major rather than the normal Th1 response that is induced in mice that have a genetically resistant background [43,44]. Lipopolysaccharide activation induced the Notch ligands Jagged1 and Delta4 on dendritic cells, and Jagged1 engagement by T cell Notch favored Th2 differentiation; T cells deficient in RBP-Jk, a key mediator of Notch Current Opinion in Immunology 2006, 18:271–277
signals, demonstrated impaired Th2 differentiation [45]. In contrast, other studies have reported that Notch has either no role [46] or positive roles in Th1 [47] or Th2 [48] development. These studies all used different approaches, and we still do not have a complete understanding of the role of Notch in Th lineage instruction. Cytokines are powerfully inductive signals, and it has been proposed that natural killer cells and basophils provide cytokines for Th1 and Th2 differentiation, respectively [49,50]. Like Th1 cells, Th2 cells are distributed widely, even after local infection, and the signals that sustain these cells in peripheral sites are unclear [51]. A selection model posits that primed T cells will express diverse cytokine receptors; it is thought that this allows preferential survival and outgrowth in distinct microenvironments. For example, IFNg activates Stat1-mediated signals that promote T-bet expression. This drives IL12Rb2 expression, in turn mediating IL-12 signaling via Stat4 to enhance IFNg induction and IL-18R expression. The result is a feed-forward loop that amplifies Th1 outgrowth when IFNg is in the environment. Novel cytokines that promote release of Th2-associated cytokines and that might operate in a similar way include thymic stromal lymphopoietin [52,53], IL-25 [54] and IL33 [55]. Insights regarding the regulation of the receptors for these cytokines will be informative, as these ultimately allow the cell to respond to their presence in the environment. An interesting wrinkle on this idea was the observation that T cell IFNg receptors, but not IL-4 receptors, were mobilized to the immunologic synapse, and that this was blocked by exogenous IL-4 [56]. Intriguingly, IFNg secretion was focused at the synapse, whereas IL-4 secretion occurred multidirectionally [57]. The question of whether this occurs in vivo will require further study [58], but the concept that cytokines might regulate the quality of dendritic cell–T cell interactions, and thus affect signal strength, is appealing. CCL21 was demonstrated to possess co-stimulatory activity during T cell priming, enhancing proliferation and IFNg production [59]. CXCL12–CXCR4 and CCL5– CCR5 were recruited into the immunologic synapse [60]. Cells that had sequestered their chemokine receptors at the synapse became refractory to chemokine gradients and formed more stable dendritic cell–T cell conjugates, presumably augmenting TCR-mediated signals.
Conclusions Th subsets underpin much of the immune response in health and disease; this accounts for the continued interest in understanding how these subsets come to be established, sustained and turned over. The discovery of additional subsets will fuel interest in identification of underlying regulatory transcription factors that are likely to be implicated in mechanisms that modify the signature cytokine genes involved in their effector function. As the www.sciencedirect.com
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‘rules’ for more of these patterns are deciphered, we will gain much understanding about fundamental developmental issues that determine cell fate decisions. Progress in mechanistic issues related to the regulation and epigenetic stabilization of cytokine genes has been matched by more sophisticated models that allow robust testing in vivo. Several laboratories are beginning to combine investigation of cytokine regulation in vivo with cell fate mapping studies, which promise to capture more fully the complexities of the working immune system.
Acknowledgements The authors thank laboratory members for helpful discussions and acknowledge support from the National Institutes of Health AI30663 and the Howard Hughes Medical Institute. RLR is a Juvenile Diabetes Research Foundation-Irvington Institute Postdoctoral Fellow. S-JK is a Cancer Research Institute Postdoctoral Fellow.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Murphy KM, Reiner SL: The lineage decisions of helper T cells. Nat Rev Immunol 2002, 2:933-944.
2.
Ansel KM, Lee DU, Rao A: An epigenetic view of helper T cell differentiation. Nat Immunol 2003, 4:616-623.
3.
Chtanova T, Tangye SG, Newton R, Frank N, Hodge MR, Rolph MS, Mackay CR: T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol 2004, 173:68-78.
4.
Vinuesa CG, Tangye SG, Moser B, Mackay CR: Follicular B helper cells in antibody responses and autoimmunity. Nat Rev Immunol 2005, 5:853-865.
5.
Hardtke S, Ohl L, Forster R: Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B cell help. Blood 2005, 106:1924-1931. The authors use adoptive transfer to demonstrate that positioning of ThFH cells in locations at the follicular borders required for the effective activation of B cells is caused by a balanced expression of CXCR5 and CCR7, which recruit T cells into B cell follicles or constrains them in the T-cell areas of the lymph node, respectively. 6.
Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, Hartley SB, O’Garra A, Cahalan MD, Cyster JG: Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol 2005, 3:e150.
7.
Ebert LM, Horn MP, Lang AB, Moser B: B cells alter the phenotype and function of follicular-homing CXCR5+ T cells. Eur J Immunol 2004, 34:3562-3571.
8.
Akiba H, Takeda K, Kojima Y, Usui Y, Harada N, Yamazaki T, Ma J, Tezuka K, Yagita H, Okumura K: The role of ICOS in the CXCR5+ follicular B helper T cell maintenance in vivo. J Immunol 2005, 175:2340-2348. The authors use ICOS blockade with anti-B7RP1 monoclonal antibody and ICOS-deficient mice to demonstrate that ICOS expression on activated T cells has a role in enhancing CXCR5 expression and a ThFH fate. 9.
Vinuesa CG, Cook MC, Angelucci C, Athanasopoulos V, Rui L, Hill KM, Yu D, Domaschenz H, Whittle B, Lambe T et al.: A RING-type unbiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 2005, 435:452-458.
10. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ: IL-23 drives www.sciencedirect.com
a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005, 201:233-240. 11. Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T, Kastelein RA, Sedgwick JD, Cua DJ: Divergent pro- and anti-inflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 2003, 198:1951-1957. 12. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, Weaver CT: Interleukin 17-producing CD4 effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005, 6:1123-1132. This study and [13] demonstrate that Th17 cells differentiate by use of signaling molecules and transcription factors distinct from those required for Th1/Th2 differentiation. This justifies the recognition of this as the third distinct lineage capable of differentiation directly from naı¨ve CD4 T cells. 13. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, Wang Y, Hood L, Zhu Z, Tian Q, Dong C: A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005, 6:1133-1141. See annotation to [12]. 14. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B: TGF-b in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006, 24:179-189. 15. Lund R, Ahlfors H, Kainonen E, Lahesmaa AM, Dixon C, Lahesmaa R: Identification of genes involved in the initiation of human Th1 or Th2 cells commitment. Eur J Immunol 2005, 35:3307-3319. 16. Kimura MY, Hosokawa H, Yamashita M, Hasegawa A, Iwamura C, Watarai H, Taniguchi M, Takagi T, Ishii S, Nakayama T: Regulation of T helper type 2 cell differentiation by murine Schnurri-2. J Exp Med 2005, 201:397-408. 17. Corn RA, Hunter C, Liou HC, Siebenlist U, Boothby MR: Opposing roles for RelB and Bcl-3 in regulation of T-box expressed in T cells, GATA-3, and Th effector differentiation. J Immunol 2005, 175:2102-2110. 18. Yamane H, Zhu J, Paul WE: Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment. J Exp Med 2005, 202:793-804. 19. Yamashita M, Shinnakasu R, Asou H, Kimura M, Hasegawa A, Hashimoto K, Hatano N, Ogata M, Nakayama T: Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. J Biol Chem 2005, 280:29409-29419. This study eloquently demonstrates that TCR-mediated activation of ERK stabilizes GATA3 protein by inhibiting the ubiquitin–proteosome pathway; this provides biochemical underpinnings for a signal-strength element in activation of IL-4 expression. 20. Hwang ES, Szabo SJ, Schwartzberg PL, Glimcher LH: T helper cell fate specified by kinase-mediated interaction of T-bet with GATA3. Science 2005, 307:430-433. The authors demonstrate that Itk phosphorylates T-bet on Tyr525 under Th1 conditions, thus facilitating an interaction between T-bet and GATA3 that diminishes binding of GATA3 to target DNA sequences. This study reveals previously unappreciated direct physical interactions between the Th subset master regulators. 21. Miller AT, Wilcox HM, Lai Z, Berg LJ: Signaling through Itk promotes T helper 2 differentiation via negative regulation of T-bet. Immunity 2004, 21:67-80. 22. Hwang ES, Hong JH, Glimcher LH: IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. J Exp Med 2005, 202:1289-1300. 23. Shinohara ML, Jansson M, Hwang ES, Werneck MB, Glimcher LH, Cantor H: T-bet-dependent expression of osteopontin contributes to T cell polarization. Proc Natl Acad Sci USA 2005, 102:17101-17106. 24. Martins GA, Hutchins AS, Reiner SL: Transcriptional activators of helper T cell fate are required for establishment but not maintenance of signature cytokine expression. J Immunol 2005, 175:5981-5985. Current Opinion in Immunology 2006, 18:271–277
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25. Pai S-Y, Truitt ML, Ho I-C: GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells. Proc Natl Acad Sci USA 2004, 101:1993-1998. These manuscripts [25,26] use mice that have a conditional GATA3 allele to demonstrate requirements for GATA3 not only in Th2 differentiation and maintenance but also in inhibition of Th1 differentiation. The use of an OX40-drive Cre system allowed the authors of [26] to achieve more efficient deletion of GATA3 for analysis in in vivo systems. 26. Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, Killeen N, Urban JF Jr, Guo L, Paul WE: Conditional deletion of Gata3 shows its essential function in T(H)1–T(H)2 responses. Nat Immunol 2004, 5:1157-1165. See annotation to [25]. 27. Chang HC, Zhang S, Thieu VT, Slee RB, Bruns HA, Laribee RN, Klemsz MJ, Kaplan MH: PU.1 expression delineates heterogeneity in primary Th2 cells. Immunity 2005, 22:693-703. 28. Guo L, Hu-Li J, Paul WE: Probabilistic regulation in Th2 cells accounts for monoallelic expression of IL-4 and IL-13. Immunity 2005, 23:89-99. 29. Fields PE, Lee GR, Kim ST, Bartsevich VV, Flavell RA: Th2specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region. Immunity 2004, 21:865-876. 30. Lee GR, Spilianakis CG, Flavell RA: Hypersensitive site 7 of the Th2 locus control region is essential for expressing Th2 cytokine genes and for long-range intrachromosomal interactions. Nat Immunol 2005, 6:42-48. 31. Lee DU, Rao A: Molecular analysis of a locus control region in the T helper 2 cytokine gene cluster: a target for Stat6 but not GATA3. Proc Natl Acad Sci USA 2004, 101:16010-16015. 32. Baguet A, Bix M: Chromatin landscape dynamics of the Il4–Il13 locus during T helper 1 and 2 development. Proc Natl Acad Sci USA 2004, 101:11410-11415. 33. Ansel KM, Greenwald RJ, Agarwal S, Bassing CH, Monticelli S, Interlandi J, Djuretic IM, Lee DU, Sharpe AH, Alt FW, Rao A: Deletion of a conserved IL4 silencer impairs T helper type 1-mediated immunity. Nat Immunol 2004, 5:1251-1259. In this study, the authors deleted a genetically conserved hypersensitivity site 350 bp long between the IL-4 coding region and the 30 -enhancer. They demonstrated that cultured Th1 cells from these mice aberrantly produce IL-4 after stimulation in vitro. The animals also failed to adequately control L. major infection owing to the increased expression of IL4 from Th cells. 34. Koyanagi M, Baguet A, Martens J, Margueron R, Jenuwein T, Bix M: EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells. J Biol Chem 2005, 280:31470-31477. 35. Baguet A, Sun X, Arroll T, Krumm A, Bix M: Intergenic transcription is not required in Th2 cells to maintain histone acetylation and transcriptional permissiveness at the il4–il13 locus. J Immunol 2005, 175:8146-8153. 36. Muljo SA, Ansel KM, Kanellopoulou C, Livingston DM, Rao A, Rajewsky K: Aberrant T cell differentiation in the absence of dicer. J Exp Med 2005, 202:261-269. 37. Shnyreva M, Weaver WM, Blanchette M, Taylor SL, Tompa M, Fitzpatrick DR, Wilson CB: Evolutionarily conserved sequence elements that positively regulate IFNg expression in T cells. Proc Natl Acad Sci USA 2004, 101:12622-12627. 38. Lee DU, Avni O, Chen L, Rao A: A distal enhancer in the interferon-g (IFNg) locus revealed by genome sequence comparison. J Biol Chem 2004, 279:4802-4810. 39. Cho JY, Grigura V, Murphy TL, Murphy K: Identification of cooperative monomeric brachyury sites conferring T-bet responsiveness to the proximal IFN-g promoter. Int Immunol 2003, 15:1149-1160. 40. Tong Y, Aune T, Boothby M: T-bet antagonizes mSin3a recruitment and transactivates a fully methylated IFN-g promoter via a conserved T-box half-site. Proc Natl Acad Sci USA 2005, 102:2034-2039. The authors demonstrate that T-bet can bind to the IFN-g promoter, even in a fully methylated state, and can transactivate gene expression in collaboration with CCAAT/enhancer binding protein b. Enforced expresCurrent Opinion in Immunology 2006, 18:271–277
sion of T-bet could override repressive elements from a chromatinized promoter, consistent with epigenetic modifications that have been previously demonstrated. 41. Spilianakis CG, Lalioti MD, Town T, Lee GR, Flavell RA: Interchromosomal associations between alternatively expressed loci. Nature 2005, 435:637-645. The authors use the powerful chromosome conformation capture (3C) technique to demonstrate physical association in naı¨ve CD4 T cells between regulatory elements in the IL-4–IL-13–IL-5 Th2 cytokine locus on chromosome 11 and the IFN-g promoter on chromosome 10. These interchromosomal interactions were dynamically lost to favor intrachromosomal reorganization upon cell activation. 42. Yarovinsky F, Zhang D, Anderson JF, Bannenberg GL, Serhan CN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A: TLR11 activation by dendritic cells by a protozoan profilinglike protein. Science 2005, 308:1626-1629. 43. Muraille E, De Trez C, Brait M, De Baetselier P, Leo O, Calier Y: Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response. J Immunol 2003, 170:4237-4241. 44. Debus A, Glasner J, Rollinghoff M, Gessner A: High levels of susceptibility and T helper 2 response in MyD88-deficient mice infected with Leishmania major are interleukin 4 dependent. Infect Immun 2003, 71:7215-7218. 45. Amsen D, Blander JM, Lee GR, Tanigaki K, Honjo T, Flavell RA: Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 2004, 117:515-526. 46. Tacchini-Cottier F, Allenbach C, Otten LA, Radtke F: Notch1 expression on T cells is not required for CD4 T helper differentiation. Eur J Immunol 2004, 34:1588-1596. 47. Minter LM, Turley DM, Das P, Shin HM, Joshi I, Lawlor RG, Cho OH, Palaga T, Gottipati S, Telfer JC et al.: Inhibitors of gamma-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat Immunol 2005, 6:680-688. 48. Tu L, Fang TC, Artis D, Shestova O, Pross SE, Maillard I, Pear WS: Notch signaling is an important regulator of type 2 immunity. J Exp Med 2005, 202:1037-1042. 49. Laouar Y, Sutterwala FA, Gorelik L, Flavell RA: Transforming growth factor b controls T helper type 1 cell development through regulation of natural killer interferon-g. Nat Immunol 2005, 6:600-607. 50. Hida S, Tadachi M, Saito T, Taki S: Negative control of basophil expression by IRF-2 critical for the regulation of Th1/Th2 balance. Blood 2005, 106:2011-2017. 51. Mohrs K, Wakil AE, Killeen N, Locksley RM, Mohrs M: A two-step process for cytokine production revealed by IL-4 dualreporter mice. Immunity 2005, 23:419-429. 52. Zhou B, Comeau MR, De Smedt T, Liggitt HD, Dahl ME, Lewis DB, Gyarmati D, Aye T, Campbell DJ, Ziegler SF: Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice. Nat Immunol 2005, 6:1047-1053. 53. Al-Shami A, Spolski R, Kelly J, Keane-Meyers A, Leonard WJ: A role for TSLP in the development of inflammation in an asthma model. J Exp Med 2005, 202:829-839. 54. Fort MM, Cheung J, Yen D, Li J, Zurawski SM, Lo S, Menon S, Clifford T, Hunte B, Lesley R et al.: IL-25 induces IL-4, IL-5 and IL-13 and Th2-associated pathologies in vivo. Immunity 2001, 15:985-995. 55. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X et al.: IL-33, an interleukin 1-like cytokine that signals via the IL-1 receptor-related ST2 and induces T helper type 2-associated cytokines. Immunity 2005, 23:479-490. 56. Maldonado RA, Irvine DJ, Schreiber R, Glimcher LH: A role for the immunological synapse in lineage commitment of CD4 lymphocytes. Nature 2004, 431:527-532. The authors demonstrate that activation of naı¨ve CD4 T cells results in recruitment of IFNg receptors into the immunological synapse. IL-4 www.sciencedirect.com
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receptors were not similarly localized and remained peripherally distributed. Remarkably, exogenous IL-4 blocked the recruitment of IFNg receptors, suggesting a physical underpinning to the ability of IL-4 to disrupt Th1 differentiation.
58. Haring JS, Badovinac VP, Olson MR, Varga SM, Harty JT: In vivo generation of pathogen-specific Th1 cells in the absence of the IFN-g receptor. J Immunol 2005, 175:3117-3122.
57. Huse M, Lillemeier BF, Kuhns MS, Chen DS, Davis MM: T cells use two directionally distinct pathways for cytokine secretion. Nat Immunol 2006, 7:247-255. These authors use imaging techniques to distinguish secretion of some cytokines, including IL-2, IFNg and IL-10, at the immunologic synapse, and others, including IL-4 and TNFa, multidirectionally and away from the synapse. This suggests that these patterned cytokines are directed at distinct cellular targets.
59. Flanagan K, Moroziewicz D, Kwak H, Horig H, Kaufman HL: The lymphoid chemokine CCL21 costimulates naı¨ve T cell expansion and Th1 polarization of non regulatory CD4 T cells. Cell Immunol 2004, 231:75-84. 60. Molon B, Gri G, Bettella M, Gomez-Mouton C, Lanzavecchia A, Martinez AC, Manes S, Viola A: T cell costimulation by chemokine receptors. Nat Immunol 2005, 6:465-471.
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Current Opinion in Immunology 2006, 18:271–277
Current Opinion in Immunology 2006, 18:271–277 This review comes from a themed issue on Lymphocyte activation Edited by Bernard Malissen and Janet Stavnezer Available online 17th April 2006 0952-7915/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2006.03.003
Introduction CD4 helper T (Th) cells orchestrate host immunity by secreting cytokines; these act on immune and nonimmune cells to regulate the response to pathogens. Naı¨ve CD4 T cells are maintained in a pluripotent state and have a relatively quiescent effector program as they recirculate from blood through lymphoid organs surveying dendritic cells for activating MHC–peptide complexes. Through complex mechanisms that integrate signals from activated dendritic cells and from the cytokine milieu, naı¨ve T cells are driven through rapid rounds of division that are linked intimately with the acquisition of the capacity to secrete effector cytokines necessary to confront distinct groups of pathogens. These events have been correlated with epigenetic changes in DNA and chromatin surrounding the cytokine genes, which reflects their competent or silenced states [1,2]. With time, daughter cells become fully differentiated and fixed in www.sciencedirect.com
their effector lineages and migrate to sites where their cytokines functionally organize the immune response. In this review, we will focus on recent studies that have identified new subsets of Th cells, new insights regarding the activation and/or silencing of discrete cytokine networks in these subsets, and investigations into the role of dendritic cells and other innate cells in regulating the differentiation, trafficking and/or maintenance of Th subsets.
The who — new Th subsets Additional Th subsets have been characterized that have effector functions distinct from the canonical Th1 (interferon [IFN]g-secreting) and Th2 (interleukin [IL]-4secreting) subsets; these include Tr1 (IL-10-secreting), Th3 (transforming growth factor [TGF]b-producing), ThFH (follicular helper cells), peripherally-induced T regulatory (Treg; FoxP3-positive) and Th17 (IL17A-producing) cells (Figure 1). Although the increasing amount of literature on Treg will not be covered in this review, new information concerning ThFH and Th17 cells has increased our understanding of these subsets and will be discussed. ThFH cells
ThFH cells, which were first identified in humans, represent T cells found in germinal centers that possess a cytokine profile distinct from Th1 and Th2 cells. ThFH cells have a different transcriptional profile compared with Th1 and Th2 cells, and selectively express CD84, CD200, B cell lymphoma (Bcl-6) and IL-2 [3,4]. Although subsets of ThFH cells have been described, we define ThFH cells to be cells that express CXCR5 and that migrate to B cell follicles after activation. T cell–B cell interactions at the follicular border are crucial for efficient B cell activation and for antibody production, and migration to the border is dependent upon the expression of chemokine receptors by both T and B cells. CXCR5 is responsible for ThFH cell migration and a recent study suggests that a push–pull mechanism positions these cells [5]. In CXCR5-deficient mice, antigen-specific T cells were retained in the lymph node T cell areas by CCR7; this suggests that competition between two chemokine receptors positions activated T cells. Imaging studies support the evidence that activated B cells modulate chemokine receptor expression and move to the follicular border in a CCR7-dependent manner, where they engage T cells and actively pull them into the Current Opinion in Immunology 2006, 18:271–277
272 Lymphocyte activation
Figure 1
T helper subsets. Although the precise lineage of the subsets in blue has not been defined by cell fate-mapping experiments, Th1, Th17 and Th2 cells represent alternative effector cell fates individually derived from naı¨ve precursor T cells (Thp). The degree to which these cell fates are conditioned by different classes of dendritic cells (DC) is unknown. The differentiation, maintenance and effector functions of these subsets share certain symmetries. Th1 cells express T-bet and IL-12 receptors (IL-12R) downstream of IFNg signals, and are further maintained by IL-12 and IL-18, members of the greater IL-6-like and IL-1-like superfamilies, respectively. Th17 cells develop in the presence of TGFb and IL-6, and are further maintained by IL-23 and IL-1b, members of the same superfamilies. Th2 cells are maintained by IL-4 itself, as well as by the IL-1 superfamily member IL-33. Downstream, Th subsets recruit distinct innate cell types to mediate effector functions in the periphery.
follicle [6]. Another study, which used isolated human ThFH cells and activated B cells, indicated that ThFHmediated B cell help could come via the B cell itself [7]. This study suggests that B cells can modulate the expression of certain costimulatory and chemokine receptors on ThFH cells, but this depends on the activation status of the B cell and on the presence of antigen. Indeed, recent literature supports the idea that multiple costimulatory molecules and their ligands on T and B cells play important roles in optimal antibody production and in class switching. Inducible costimulatory molecule (ICOS) is one such molecule. ICOS-deficient mice or mice treated with an antibody against its receptor had a reduced number of ThFH cells [8]. CXCR5 expression on CD4 T cells was linked to ICOS expression and was enhanced by interactions with B cells. CD4 T cells from mice that have a mutated roquin gene expressed high levels of ICOS and these animals had elevated numbers of ThFH cells [9]. These mice also had abnormally high numbers of germinal centers and eventually developed autoimmune disease, which highlights the potential importance of ThFH cells in regulating autoantibody production. As both IL-4-secreting and IFNg-secreting Th cells have been identified in follicles, the precise lineage of ThFH cells as a unique or terminally differentiated state of Th subset development remains unclear. Current Opinion in Immunology 2006, 18:271–277
Th17 cells
Th17 cells have been linked to model autoimmune diseases including experimental allergic encephalomyelitis and collagen-induced arthritis [10,11]. Th17 cells secrete IL-17A, IL-17F, IL-6 and tumor necrosis factor (TNF)a, and their functions reflect the capacities of these cytokines collectively to mobilize, to recruit and to activate neutrophils, in part by induction of chemokines and growth factors from diverse cell types. Two recent studies [12,13] support a hypothesis wherein these cells arise as a separate Th lineage, and predict that they do not represent a population of Th1 cells that have undergone further differentiation. When naı¨ve CD4 T cells were cultured in the presence of antiIFNg during priming, a large percentage of the cells generated were Th17 cells. An additive effect was observed when IL-4 was blocked, which implies that the development of Th17 cells is inhibited by the presence of IFNg and IL-4. Further evidence that these are a unique lineage of Th cells came from experiments that showed that cells from mice deficient in T-box expressed in T cells (T-bet), signal transducer and activator of transcription 4 (Stat4) or Stat6 had no defect in IL-17 production. Thus, traditional regulators of Th1 and Th2 cells did not appear to be www.sciencedirect.com
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necessary for Th17 differentiation. The transcription factors T-bet and GATA-binding protein 3 (GATA3), which have been associated with Th1 and Th2 differentiation, respectively, were not required for Th17 development in vivo [14]. Interestingly, this study showed that TGFb produced by CD4 Treg cells in the presence of IL-6 was responsible for Th17 differentiation, and that the further addition of IL-1b or TNFa led to highly efficient differentiation of these cells. Although not required for their development, IL-23 was shown to be important in the survival and maintenance of Th17 cells. Lund et al. [15] found Th1 and Th2 mRNA expression profiles were altered when naı¨ve cells were cultured in the presence of TGFb. It will be interesting to see if any of the 20 genes that they determined to be regulated by TGFb might be a transcription factor that acts as a ‘master regulator’ for the Th17 lineage.
The how – signal integration, feed-forward amplification and crosstalk Signal strenth
Activation of alternative cytokine fates during naı¨ve CD4 T cell priming has been attributed to alterations in the ‘strength of signal’ layered onto extensive cross-regulation between the lineages. Accumulating evidence has implicated the nuclear transcription factor (NF)-kB and extracellular signal-related kinase (ERK) cascades in modulating signal strength. Schnurri-2 is a transcriptional repressor that competes with p50 for binding to consensus NF-kB motifs. Schnurri-2-deficient T cells demonstrated constitutive NF-kB activation and enhanced Th2 differentiation, which correlated with increased induction of GATA3 [16]. Conversely, T cells deficient in the NF-kB family member Bcl-3 had decreased GATA3 induction and Th2 differentiation, perhaps reflecting the ability of the Bcl-3–p50 complex to bind and transactivate the GATA3 gene in in vitro assays [17]. It will be interesting to see whether Schnurri-2 represses the GATA3 promoter by antagonizing Bcl-3–p50 complexes in Th2 cells. Cells from mice deficient in the NF-kB family member RelB demonstrated impaired Th1 differentiation, which correlated with deficiencies in T-bet and Stat4 induction after activation. Transient ERK activation by weak TCR–CD28 signals induced IL-2R-dependent Stat5 phosphorylation, GATA3 expression and IL-4 production; these were abolished by stimulation with high antigen doses that induced sustained ERK activation [18]. Pharmacologic ERK inhibitors decreased GATA3 protein levels in developing Th2 cells by enhanced ubiquitin–proteosome degradation. ERK activation stabilized GATA3 protein levels, and Mdm2 could act as an E3 ligase for GATA3 [19]. Further work is needed to assess whether ERKmitogen activated protein kinase (MAPK)-mediated phosphorylation of GATA3 is responsible for these effects. ERK signals also mediate GATA3-dependent www.sciencedirect.com
histone H3 acetylation of Th2 cytokine loci, suggesting that this pathway influences GATA3 function at multiple levels. T-bet versus GATA3
One observation in the T-bet knockout mouse was the tendency of animals to spontaneously develop Th2mediated inflammation. During Th1 priming, T-bet was phosphorylated on Tyr525 by the Tec kinase Itk. Although Tyr525 phosphorylation of T-bet did not affect activation of IFNg, this modification facilitated a physical interaction of T-bet with GATA3, resulting in its sequestration from binding sites in the Th2 cytokine locus [20]. At low levels of TCR–CD28 activation, however, Itk was required for efficient GATA3 induction and for Th2 development [21], indicating that further complexities underlie the crosstalk between these master regulators. An additional modification of T-bet involved Ser508 phosphorylation by casein kinase I and glycogen synthase kinase-3. This modification led to attenuation of IL-2 production as it promoted heterodimers of T-bet and RelA, thus interfering with RelA activation of the IL-2 promoter [22]. Additional targets of T-bet continued to be identified. Osteopontin is a T-bet-dependent gene in Th1 cells [23], and the transcription factor Hlx is a Tbet-induced gene that interacts physically with T-bet in a feed-forward mechanism required for optimal activation, but not maintenance, of the IFNg locus [24]. Conditional GATA3 knockout cells were used to demonstrate the non-redundant role for GATA3 in both initiating and maintaining Th2 cell fate [25,26]. GATA3 function could be attenuated by PU.1, an ETS family transcription factor implicated in myeloid and B cell fate decisions, which was induced transiently in a Stat6dependent manner after Th2 priming [27]. Intriguingly, PU.1 induction was variegated in the population and interfered with GATA3 DNA binding; it is possible that this contributed to allelic diversity in the spectrum of expressed Th2-family cytokines [28]. Sequence mining
Novel regulatory elements continue to be revealed by mining sequences across the Th1 and Th2 cytokine loci. A major finding was the identification of a rad50embedded element upstream of IL-13 and IL-4; this functioned as a locus control region that had both enhancer and chromatin-modifying activity in mediating Th2 cytokine expression by long-range intrachromosomal interactions [29,30]. This locus control region was demonstrated to undergo indiscriminate modification after T cell priming, although an additional DNase I hypersensitivity site developed in response to Stat6-mediated signals [31]. Dynamic mapping of chromatin alterations across the Th2 locus indicated that DNase hypersensitivity site IV, which is 30 of Il4, is a repressive element [32]; this was confirmed by targeted deletion [33]. Th1 Current Opinion in Immunology 2006, 18:271–277
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cells from silencer-deficient mice continued to produce IL-4, and were unable to use efficient Th1-mediated immunity to clear pathogens such as Leishmania major. Silencing in the Th2 locus of Th1 cells was associated with methylated H3 accumulation mediated by the Polycomb family methyltransferase EZH2 [34]. Intriguingly, intergenic transcription could be detected in the silenced Th2 locus, raising the possibility that RNA-based silencing mechanisms might occur [35]. Initial evaluation of Dicer-deficient CD4 cells revealed inappropriate IFNg production during Th differentiation [36]. Progress has also been made in identifying elements that regulate activation of the Ifng gene. Enhancer elements 5 kb upstream and 18 kb downstream of the gene underwent histone modifications during Th1 priming [37], and the upstream element was demonstrated to bind NF-AT and T-bet in chromatin immunoprecipitations [38]. T-bet was demonstrated to bind directly at the Ifng promoter utilizing conserved T-box half-sites to transactivate the gene in cooperation with CCAAT/ enhancer binding protein b [39,40]. Enforced T-bet expression could dissociate the mSin3a co-repressor from the endogenous locus, consistent with the capacity of Tbet to transactivate the Ifng gene when introduced into Th2 cells. A striking finding was an interchromosomal association between regulatory elements of the Th2 cytokine locus on chromosome 11 and the Ifng promoter on chromosome 10 in the mouse. Th differentiation was correlated with chromosomal reorganization that favored intrachromosomal interactions, suggesting that this physically underpins alternative cytokine expression patterns [41]. It would be interesting to consider these interactions in natural killer T cells — an atypical lineage that co-expresses IFNg and IL-4.
The where — instruction or selection? Interest continues to surround the issue of how differentiation of Th substrates is regulated: is this controlled by dendritic cells conditioned in response to distinct pathogens or is there selection by a more stochastically regulated process by which primed T cells respond to cytokines in the environment, which promote their subsequent survival and growth. Activation of a TLR11dependent pathway in dendritic cells by a Toxoplasma profilin-like molecule led to IL-12 induction, which potentially accounts for the strong Th1 response to infection by this organism [42]. Conversely, MyD88-deficient mice developed aberrant Th2 responses to L. major rather than the normal Th1 response that is induced in mice that have a genetically resistant background [43,44]. Lipopolysaccharide activation induced the Notch ligands Jagged1 and Delta4 on dendritic cells, and Jagged1 engagement by T cell Notch favored Th2 differentiation; T cells deficient in RBP-Jk, a key mediator of Notch Current Opinion in Immunology 2006, 18:271–277
signals, demonstrated impaired Th2 differentiation [45]. In contrast, other studies have reported that Notch has either no role [46] or positive roles in Th1 [47] or Th2 [48] development. These studies all used different approaches, and we still do not have a complete understanding of the role of Notch in Th lineage instruction. Cytokines are powerfully inductive signals, and it has been proposed that natural killer cells and basophils provide cytokines for Th1 and Th2 differentiation, respectively [49,50]. Like Th1 cells, Th2 cells are distributed widely, even after local infection, and the signals that sustain these cells in peripheral sites are unclear [51]. A selection model posits that primed T cells will express diverse cytokine receptors; it is thought that this allows preferential survival and outgrowth in distinct microenvironments. For example, IFNg activates Stat1-mediated signals that promote T-bet expression. This drives IL12Rb2 expression, in turn mediating IL-12 signaling via Stat4 to enhance IFNg induction and IL-18R expression. The result is a feed-forward loop that amplifies Th1 outgrowth when IFNg is in the environment. Novel cytokines that promote release of Th2-associated cytokines and that might operate in a similar way include thymic stromal lymphopoietin [52,53], IL-25 [54] and IL33 [55]. Insights regarding the regulation of the receptors for these cytokines will be informative, as these ultimately allow the cell to respond to their presence in the environment. An interesting wrinkle on this idea was the observation that T cell IFNg receptors, but not IL-4 receptors, were mobilized to the immunologic synapse, and that this was blocked by exogenous IL-4 [56]. Intriguingly, IFNg secretion was focused at the synapse, whereas IL-4 secretion occurred multidirectionally [57]. The question of whether this occurs in vivo will require further study [58], but the concept that cytokines might regulate the quality of dendritic cell–T cell interactions, and thus affect signal strength, is appealing. CCL21 was demonstrated to possess co-stimulatory activity during T cell priming, enhancing proliferation and IFNg production [59]. CXCL12–CXCR4 and CCL5– CCR5 were recruited into the immunologic synapse [60]. Cells that had sequestered their chemokine receptors at the synapse became refractory to chemokine gradients and formed more stable dendritic cell–T cell conjugates, presumably augmenting TCR-mediated signals.
Conclusions Th subsets underpin much of the immune response in health and disease; this accounts for the continued interest in understanding how these subsets come to be established, sustained and turned over. The discovery of additional subsets will fuel interest in identification of underlying regulatory transcription factors that are likely to be implicated in mechanisms that modify the signature cytokine genes involved in their effector function. As the www.sciencedirect.com
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‘rules’ for more of these patterns are deciphered, we will gain much understanding about fundamental developmental issues that determine cell fate decisions. Progress in mechanistic issues related to the regulation and epigenetic stabilization of cytokine genes has been matched by more sophisticated models that allow robust testing in vivo. Several laboratories are beginning to combine investigation of cytokine regulation in vivo with cell fate mapping studies, which promise to capture more fully the complexities of the working immune system.
Acknowledgements The authors thank laboratory members for helpful discussions and acknowledge support from the National Institutes of Health AI30663 and the Howard Hughes Medical Institute. RLR is a Juvenile Diabetes Research Foundation-Irvington Institute Postdoctoral Fellow. S-JK is a Cancer Research Institute Postdoctoral Fellow.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Murphy KM, Reiner SL: The lineage decisions of helper T cells. Nat Rev Immunol 2002, 2:933-944.
2.
Ansel KM, Lee DU, Rao A: An epigenetic view of helper T cell differentiation. Nat Immunol 2003, 4:616-623.
3.
Chtanova T, Tangye SG, Newton R, Frank N, Hodge MR, Rolph MS, Mackay CR: T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol 2004, 173:68-78.
4.
Vinuesa CG, Tangye SG, Moser B, Mackay CR: Follicular B helper cells in antibody responses and autoimmunity. Nat Rev Immunol 2005, 5:853-865.
5.
Hardtke S, Ohl L, Forster R: Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B cell help. Blood 2005, 106:1924-1931. The authors use adoptive transfer to demonstrate that positioning of ThFH cells in locations at the follicular borders required for the effective activation of B cells is caused by a balanced expression of CXCR5 and CCR7, which recruit T cells into B cell follicles or constrains them in the T-cell areas of the lymph node, respectively. 6.
Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, Hartley SB, O’Garra A, Cahalan MD, Cyster JG: Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol 2005, 3:e150.
7.
Ebert LM, Horn MP, Lang AB, Moser B: B cells alter the phenotype and function of follicular-homing CXCR5+ T cells. Eur J Immunol 2004, 34:3562-3571.
8.
Akiba H, Takeda K, Kojima Y, Usui Y, Harada N, Yamazaki T, Ma J, Tezuka K, Yagita H, Okumura K: The role of ICOS in the CXCR5+ follicular B helper T cell maintenance in vivo. J Immunol 2005, 175:2340-2348. The authors use ICOS blockade with anti-B7RP1 monoclonal antibody and ICOS-deficient mice to demonstrate that ICOS expression on activated T cells has a role in enhancing CXCR5 expression and a ThFH fate. 9.
Vinuesa CG, Cook MC, Angelucci C, Athanasopoulos V, Rui L, Hill KM, Yu D, Domaschenz H, Whittle B, Lambe T et al.: A RING-type unbiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 2005, 435:452-458.
10. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ: IL-23 drives www.sciencedirect.com
a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 2005, 201:233-240. 11. Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T, Kastelein RA, Sedgwick JD, Cua DJ: Divergent pro- and anti-inflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 2003, 198:1951-1957. 12. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, Weaver CT: Interleukin 17-producing CD4 effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005, 6:1123-1132. This study and [13] demonstrate that Th17 cells differentiate by use of signaling molecules and transcription factors distinct from those required for Th1/Th2 differentiation. This justifies the recognition of this as the third distinct lineage capable of differentiation directly from naı¨ve CD4 T cells. 13. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, Wang Y, Hood L, Zhu Z, Tian Q, Dong C: A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005, 6:1133-1141. See annotation to [12]. 14. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B: TGF-b in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006, 24:179-189. 15. Lund R, Ahlfors H, Kainonen E, Lahesmaa AM, Dixon C, Lahesmaa R: Identification of genes involved in the initiation of human Th1 or Th2 cells commitment. Eur J Immunol 2005, 35:3307-3319. 16. Kimura MY, Hosokawa H, Yamashita M, Hasegawa A, Iwamura C, Watarai H, Taniguchi M, Takagi T, Ishii S, Nakayama T: Regulation of T helper type 2 cell differentiation by murine Schnurri-2. J Exp Med 2005, 201:397-408. 17. Corn RA, Hunter C, Liou HC, Siebenlist U, Boothby MR: Opposing roles for RelB and Bcl-3 in regulation of T-box expressed in T cells, GATA-3, and Th effector differentiation. J Immunol 2005, 175:2102-2110. 18. Yamane H, Zhu J, Paul WE: Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment. J Exp Med 2005, 202:793-804. 19. Yamashita M, Shinnakasu R, Asou H, Kimura M, Hasegawa A, Hashimoto K, Hatano N, Ogata M, Nakayama T: Ras-ERK MAPK cascade regulates GATA3 stability and Th2 differentiation through ubiquitin-proteasome pathway. J Biol Chem 2005, 280:29409-29419. This study eloquently demonstrates that TCR-mediated activation of ERK stabilizes GATA3 protein by inhibiting the ubiquitin–proteosome pathway; this provides biochemical underpinnings for a signal-strength element in activation of IL-4 expression. 20. Hwang ES, Szabo SJ, Schwartzberg PL, Glimcher LH: T helper cell fate specified by kinase-mediated interaction of T-bet with GATA3. Science 2005, 307:430-433. The authors demonstrate that Itk phosphorylates T-bet on Tyr525 under Th1 conditions, thus facilitating an interaction between T-bet and GATA3 that diminishes binding of GATA3 to target DNA sequences. This study reveals previously unappreciated direct physical interactions between the Th subset master regulators. 21. Miller AT, Wilcox HM, Lai Z, Berg LJ: Signaling through Itk promotes T helper 2 differentiation via negative regulation of T-bet. Immunity 2004, 21:67-80. 22. Hwang ES, Hong JH, Glimcher LH: IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. J Exp Med 2005, 202:1289-1300. 23. Shinohara ML, Jansson M, Hwang ES, Werneck MB, Glimcher LH, Cantor H: T-bet-dependent expression of osteopontin contributes to T cell polarization. Proc Natl Acad Sci USA 2005, 102:17101-17106. 24. Martins GA, Hutchins AS, Reiner SL: Transcriptional activators of helper T cell fate are required for establishment but not maintenance of signature cytokine expression. J Immunol 2005, 175:5981-5985. Current Opinion in Immunology 2006, 18:271–277
276 Lymphocyte activation
25. Pai S-Y, Truitt ML, Ho I-C: GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells. Proc Natl Acad Sci USA 2004, 101:1993-1998. These manuscripts [25,26] use mice that have a conditional GATA3 allele to demonstrate requirements for GATA3 not only in Th2 differentiation and maintenance but also in inhibition of Th1 differentiation. The use of an OX40-drive Cre system allowed the authors of [26] to achieve more efficient deletion of GATA3 for analysis in in vivo systems. 26. Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, Killeen N, Urban JF Jr, Guo L, Paul WE: Conditional deletion of Gata3 shows its essential function in T(H)1–T(H)2 responses. Nat Immunol 2004, 5:1157-1165. See annotation to [25]. 27. Chang HC, Zhang S, Thieu VT, Slee RB, Bruns HA, Laribee RN, Klemsz MJ, Kaplan MH: PU.1 expression delineates heterogeneity in primary Th2 cells. Immunity 2005, 22:693-703. 28. Guo L, Hu-Li J, Paul WE: Probabilistic regulation in Th2 cells accounts for monoallelic expression of IL-4 and IL-13. Immunity 2005, 23:89-99. 29. Fields PE, Lee GR, Kim ST, Bartsevich VV, Flavell RA: Th2specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region. Immunity 2004, 21:865-876. 30. Lee GR, Spilianakis CG, Flavell RA: Hypersensitive site 7 of the Th2 locus control region is essential for expressing Th2 cytokine genes and for long-range intrachromosomal interactions. Nat Immunol 2005, 6:42-48. 31. Lee DU, Rao A: Molecular analysis of a locus control region in the T helper 2 cytokine gene cluster: a target for Stat6 but not GATA3. Proc Natl Acad Sci USA 2004, 101:16010-16015. 32. Baguet A, Bix M: Chromatin landscape dynamics of the Il4–Il13 locus during T helper 1 and 2 development. Proc Natl Acad Sci USA 2004, 101:11410-11415. 33. Ansel KM, Greenwald RJ, Agarwal S, Bassing CH, Monticelli S, Interlandi J, Djuretic IM, Lee DU, Sharpe AH, Alt FW, Rao A: Deletion of a conserved IL4 silencer impairs T helper type 1-mediated immunity. Nat Immunol 2004, 5:1251-1259. In this study, the authors deleted a genetically conserved hypersensitivity site 350 bp long between the IL-4 coding region and the 30 -enhancer. They demonstrated that cultured Th1 cells from these mice aberrantly produce IL-4 after stimulation in vitro. The animals also failed to adequately control L. major infection owing to the increased expression of IL4 from Th cells. 34. Koyanagi M, Baguet A, Martens J, Margueron R, Jenuwein T, Bix M: EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in Th1 cells. J Biol Chem 2005, 280:31470-31477. 35. Baguet A, Sun X, Arroll T, Krumm A, Bix M: Intergenic transcription is not required in Th2 cells to maintain histone acetylation and transcriptional permissiveness at the il4–il13 locus. J Immunol 2005, 175:8146-8153. 36. Muljo SA, Ansel KM, Kanellopoulou C, Livingston DM, Rao A, Rajewsky K: Aberrant T cell differentiation in the absence of dicer. J Exp Med 2005, 202:261-269. 37. Shnyreva M, Weaver WM, Blanchette M, Taylor SL, Tompa M, Fitzpatrick DR, Wilson CB: Evolutionarily conserved sequence elements that positively regulate IFNg expression in T cells. Proc Natl Acad Sci USA 2004, 101:12622-12627. 38. Lee DU, Avni O, Chen L, Rao A: A distal enhancer in the interferon-g (IFNg) locus revealed by genome sequence comparison. J Biol Chem 2004, 279:4802-4810. 39. Cho JY, Grigura V, Murphy TL, Murphy K: Identification of cooperative monomeric brachyury sites conferring T-bet responsiveness to the proximal IFN-g promoter. Int Immunol 2003, 15:1149-1160. 40. Tong Y, Aune T, Boothby M: T-bet antagonizes mSin3a recruitment and transactivates a fully methylated IFN-g promoter via a conserved T-box half-site. Proc Natl Acad Sci USA 2005, 102:2034-2039. The authors demonstrate that T-bet can bind to the IFN-g promoter, even in a fully methylated state, and can transactivate gene expression in collaboration with CCAAT/enhancer binding protein b. Enforced expresCurrent Opinion in Immunology 2006, 18:271–277
sion of T-bet could override repressive elements from a chromatinized promoter, consistent with epigenetic modifications that have been previously demonstrated. 41. Spilianakis CG, Lalioti MD, Town T, Lee GR, Flavell RA: Interchromosomal associations between alternatively expressed loci. Nature 2005, 435:637-645. The authors use the powerful chromosome conformation capture (3C) technique to demonstrate physical association in naı¨ve CD4 T cells between regulatory elements in the IL-4–IL-13–IL-5 Th2 cytokine locus on chromosome 11 and the IFN-g promoter on chromosome 10. These interchromosomal interactions were dynamically lost to favor intrachromosomal reorganization upon cell activation. 42. Yarovinsky F, Zhang D, Anderson JF, Bannenberg GL, Serhan CN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A: TLR11 activation by dendritic cells by a protozoan profilinglike protein. Science 2005, 308:1626-1629. 43. Muraille E, De Trez C, Brait M, De Baetselier P, Leo O, Calier Y: Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response. J Immunol 2003, 170:4237-4241. 44. Debus A, Glasner J, Rollinghoff M, Gessner A: High levels of susceptibility and T helper 2 response in MyD88-deficient mice infected with Leishmania major are interleukin 4 dependent. Infect Immun 2003, 71:7215-7218. 45. Amsen D, Blander JM, Lee GR, Tanigaki K, Honjo T, Flavell RA: Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 2004, 117:515-526. 46. Tacchini-Cottier F, Allenbach C, Otten LA, Radtke F: Notch1 expression on T cells is not required for CD4 T helper differentiation. Eur J Immunol 2004, 34:1588-1596. 47. Minter LM, Turley DM, Das P, Shin HM, Joshi I, Lawlor RG, Cho OH, Palaga T, Gottipati S, Telfer JC et al.: Inhibitors of gamma-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat Immunol 2005, 6:680-688. 48. Tu L, Fang TC, Artis D, Shestova O, Pross SE, Maillard I, Pear WS: Notch signaling is an important regulator of type 2 immunity. J Exp Med 2005, 202:1037-1042. 49. Laouar Y, Sutterwala FA, Gorelik L, Flavell RA: Transforming growth factor b controls T helper type 1 cell development through regulation of natural killer interferon-g. Nat Immunol 2005, 6:600-607. 50. Hida S, Tadachi M, Saito T, Taki S: Negative control of basophil expression by IRF-2 critical for the regulation of Th1/Th2 balance. Blood 2005, 106:2011-2017. 51. Mohrs K, Wakil AE, Killeen N, Locksley RM, Mohrs M: A two-step process for cytokine production revealed by IL-4 dualreporter mice. Immunity 2005, 23:419-429. 52. Zhou B, Comeau MR, De Smedt T, Liggitt HD, Dahl ME, Lewis DB, Gyarmati D, Aye T, Campbell DJ, Ziegler SF: Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice. Nat Immunol 2005, 6:1047-1053. 53. Al-Shami A, Spolski R, Kelly J, Keane-Meyers A, Leonard WJ: A role for TSLP in the development of inflammation in an asthma model. J Exp Med 2005, 202:829-839. 54. Fort MM, Cheung J, Yen D, Li J, Zurawski SM, Lo S, Menon S, Clifford T, Hunte B, Lesley R et al.: IL-25 induces IL-4, IL-5 and IL-13 and Th2-associated pathologies in vivo. Immunity 2001, 15:985-995. 55. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, Zurawski G, Moshrefi M, Qin J, Li X et al.: IL-33, an interleukin 1-like cytokine that signals via the IL-1 receptor-related ST2 and induces T helper type 2-associated cytokines. Immunity 2005, 23:479-490. 56. Maldonado RA, Irvine DJ, Schreiber R, Glimcher LH: A role for the immunological synapse in lineage commitment of CD4 lymphocytes. Nature 2004, 431:527-532. The authors demonstrate that activation of naı¨ve CD4 T cells results in recruitment of IFNg receptors into the immunological synapse. IL-4 www.sciencedirect.com
T helper cell effector fates Reinhardt et al. 277
receptors were not similarly localized and remained peripherally distributed. Remarkably, exogenous IL-4 blocked the recruitment of IFNg receptors, suggesting a physical underpinning to the ability of IL-4 to disrupt Th1 differentiation.
58. Haring JS, Badovinac VP, Olson MR, Varga SM, Harty JT: In vivo generation of pathogen-specific Th1 cells in the absence of the IFN-g receptor. J Immunol 2005, 175:3117-3122.
57. Huse M, Lillemeier BF, Kuhns MS, Chen DS, Davis MM: T cells use two directionally distinct pathways for cytokine secretion. Nat Immunol 2006, 7:247-255. These authors use imaging techniques to distinguish secretion of some cytokines, including IL-2, IFNg and IL-10, at the immunologic synapse, and others, including IL-4 and TNFa, multidirectionally and away from the synapse. This suggests that these patterned cytokines are directed at distinct cellular targets.
59. Flanagan K, Moroziewicz D, Kwak H, Horig H, Kaufman HL: The lymphoid chemokine CCL21 costimulates naı¨ve T cell expansion and Th1 polarization of non regulatory CD4 T cells. Cell Immunol 2004, 231:75-84. 60. Molon B, Gri G, Bettella M, Gomez-Mouton C, Lanzavecchia A, Martinez AC, Manes S, Viola A: T cell costimulation by chemokine receptors. Nat Immunol 2005, 6:465-471.
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