Th2 Cells: Orchestrating Barrier Immunity

advances in immunology, vol. 83

Th2 Cells: Orchestrating Barrier Immunity DANIEL B. STETSON, DAVID VOEHRINGER, JANE L. GROGAN, MIN XU, R. LEE REINHARDT, STEFANIE SCHEU, BEN L. KELLY, AND RICHARD M. LOCKSLEY Howard Hughes Medical Institute University of California San Francisco San Francisco, California 94143

I. Introduction: History and Definitions

In the mid- to late-1980s, Mosmann and Coffman described the division of antigen-specific murine CD4 T cell lines and clones into stable subsets based on the patterns of cytokines they produced (Mosmann and Coffman, 1989). Although the cells shared an identical surface phenotype, Th1 cells transcribed and secreted interferon-g (IFNg), IL-2, and lymphotoxin after activation, whereas Th2 cells transcribed IL-4, IL-5, and IL-13 (called p600 at that time). Voluminous contributions over the past 15 years have established that the basic underlying principles extend to both mouse and human CD4 T cells. A fundamental concept, based largely on differentiation of naive, TCR transgenic, T cells in vitro, is the capacity to differentiate a given T cell to either a Th1 or Th2 cell, depending on the stimulation milieu. Although a variety of manipulations, generally characterized as ‘‘strength of signal,’’ influence the ease with which Th1 or Th2 fates can be generated, key cytokines and cytokine receptor pathways remain the major determinants of cell fate. Th1 and Th2 cells are commonly referred to in association with ‘‘cellular’’ and ‘‘humoral’’ immunity, respectively. The role for IFNg, the cardinal cytokine of Th1 biology, in isotype switching for opsonizing antibodies, and the activation of many cell types by IL-4, the cardinal cytokine of Th2 biology, renders these appellations imprecise. Here, we refer to type 1 immunity as an immune response centrally orchestrated by Th1 cells that stably secrete IFNg. In general, the coordinated activation of phagocytes, production of opsonizing antibodies, and induction of cytolytic T cells by Th1 cells collectively describes the response to systemic invasion. In contrast, we refer to type 2 immunity as an immune response centrally orchestrated by Th2 cells that stably secrete IL-4. As such, the coordinated concentration of activated eosinophils, mast cells, and basophils by Th2 cells collectively describes the response to barrier invasion. This chapter focuses on recent information relative to the generation and activation of IL-4-expressing Th2 cells, and, where appropriate, on comparisons with other IL-4-expressing cells in the coordination of type 2 immunity. 163 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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II. Activation of IL-4 Expression in Naive CD4 T Cells

A substantial amount of information has accumulated addressing the mechanisms by which naive helper T cells activate IL-4 expression (Murphy and Reiner, 2002). The emerging model is one of lineage differentiation, by which Th2 cells establish a distinct gene expression pattern linked intimately with epigenetically stable alterations in DNA and chromatin (Smale and Fisher, 2002). With the exception of NK T cells, which will be considered later, there is little evidence for activation of IL-4 gene expression during T cell development in the thymus. Activation via the TCR is integrated with co-stimulatory and cytokine signals delivered from dendritic cells and the environmental milieu to establish IL-4 transcription in naive CD4 T cells. Assays correlated with general transcriptional competence suggest that basal modifications of chromatin impede gene expression in naive T cells, as demonstrated by the absence of DNase I hypersensitivity sites around the IL-4/IL-13 genes (Agarwal and Rao, 1998; Santangelo et al., 2002; Takemoto et al., 1998), hypoacetylation of histones (Avni et al., 2002; Bird et al., 1998; Fields et al., 2002; Valapour et al., 2002), and methylation at CpG sites across the locus (Agarwal and Rao, 1998; Bird et al., 1998; Lee et al., 2002; Santangelo et al., 2002). Genetic evidence suggests that repression is maintained by MeCP1 complexes, including MBD2 and NURD proteins, tightly associated with the locus (Hutchins et al., 2002). Activated T cells from mice deficient in MBD2, a methyl-CpG-binding protein, or in Dnmt1, a maintenance methyltransferase, ectopically express cytokines, including IL-4, consistent with a requirement for basal chromatin-mediated barriers to transcription (Hutchins et al., 2002; Lee et al., 2001b). Differential display was used to identify GATA-3, a zinc-finger transcription factor, as a highly polarized transcript expressed in Th2, but not Th1, cells (Zhang et al., 1997; Zheng and Flavell, 1997). GATA-3 is essential for normal hematopoiesis and nervous system development, but also for T cell development, and will require a conditionally mutant allele for definitive genetic analysis of its role in Th2 differentiation. Nevertheless, overexpression, silencing, and dominant negative strategies have together established a key role for GATA-3 in Th2 differentiation. GATA-3 lies genetically upstream of the appearance of chromatin modifications identified by DNase I hypersensitivity analysis (Ouyang et al., 2000). A related transcription factor, GATA-4, has the ability to decompact condensed chromatin, a key early step in the induction of albumin expression by fetal liver, using similar zinc finger motifs that have been implicated in GATA-3 chromatin remodeling (Cirillo et al., 2002; Takemoto et al., 2002). GATA-3 binding sites extend throughout the type 2 cytokine locus encompassing IL-4, IL-13, and IL-5, and, importantly, are present within sequences corresponding to the DNase hypersensitivity regions

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that develop upon Th2 polarization. Assembly of these various sequences with the IL-4 gene as a mini-locus transgene was used to demonstrate the ability of GATA-3 to enhance markedly IL-4 expression within a chromatin context that included these presumptive regulatory regions (Lee et al., 2001a). The emerging model suggests a role for GATA-3 as a ‘‘pioneer’’ factor, likely within the context of as yet unknown additional proteins, to integrate signals emanating from the T cell activation synapse and to engage and decondense chromatin at the type 2 cytokine locus. Possibly, GATA-3 initiates decompaction by displacing repressive MeCP1 complexes, thus initiating a process by which strand methylation could be rapidly diluted during processive cell division, allowing establishment of a permissive histone code. Although definitive data is lacking, several pieces of evidence favor such a model. First, GATA3 protein is present in naive CD4 T cells, and thus available for targeting immediately after T cell activation. Second, the IL-4 genes are positioned in areas of euchromatin in naive T cells, apart from highly compacted heterochromatin (Grogan et al., 2001). Third, chromatin immunoprecipitations reveal small amounts of GATA-3 bound at IL-4 regulatory elements in resting, naive T cells (Avni et al., 2002). This observation, together with the finding that IL-4 is transcribed within 30 minutes of T cell activation in a Stat6-independent manner (Grogan et al., 2001), raises the possibility that the IL-4 gene is ‘‘poised’’ for rapid transcription by intermittent stochastic interactions of GATA-3 with the locus. Spontaneous fluidity of nucleosome structure between relatively condensed and permissive states (Narlikar et al., 2001) might reconcile the apparent contradictions inherent between a chromatin-inaccessible state and the ease with which transcription can be initiated at the gene. The frequent co-expression of IL-4, IL-13, and IL-5 from single alleles in Th2 cells (Kelly and Locksley, 2000), together with their genomic co-localization, suggested that conserved cis-acting elements likely contributed to transcription. Indeed, human transgenes containing IL-4, IL-13, and IL-5 were expressed in an appropriate cell- and stimulation-dependent context in mice (Lacy et al., 2000). Comparative sequence analysis across a number of species revealed intergenic sequences, designated CNS-1 and CNS-2 (conserved noncoding sequences), localized to the 50 IL-4/IL-13 intergenic and the 30 IL-4 regions, respectively (Loots et al., 2000). Deletions of either CNS-1 or CNS-2 attenuated IL-4 expression (Mohrs et al., 2001a; Solymar et al., 2002). Intriguingly, these areas map closely with DNase I hypersensitivity sites that appear early after Th2 polarization of naive T cells (Fig. 1). GATA-3 binding sites are located within these regions, supporting the concept of early targeting of highly conserved cis-regulatory elements on either side of the IL-4 gene that serve to recruit chromatin-modifying enzymes, thus configuring the locus in a manner competent for interactions with additional DNA-modifying protein complexes. More extensive analysis defined additional conserved regulatory

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Fig 1 Regulatory elements surrounding the IL4 gene. DNAse I hypersensitivity sites (HSS, arrows) correlate closely with regions known to regulate IL-4 expression. Enhancer elements are indicated in green, and a silencer element is depicted in red. IL-4 exons are numbered and depicted as black boxes. The relative positions of each element are not drawn to scale.

elements within the promoter and the second intronic regions, and yet more distant elements likely contribute to optimal IL-4 expression. Transgenes containing each of these regulatory elements together conferred cell- and condition-specificity upon IL-4 expression in lymphocytes (Lee et al., 2001a) (Fig. 1). Although GATA-3 ‘‘brackets’’ the IL-4 gene and does not directly transactivate the IL-4 promoter, GATA-3 binds and transactivates the IL-5 and IL-13 proximal promoters directly (Kishikawa et al., 2001; Yamashita et al., 2002; Zhang et al., 1997). Both IL-4 and IL-13 bind to the widely distributed IL4Ra/IL-13Ra1 receptor, but only IL-4 binds to a lymphoid-restricted receptor, IL-4Ra/gc, leading to speculation that evolution has scaffolded lymphocytespecific regulation onto an IL-4 gene duplicated from related genes more directly activated by GATA-3 in nonlymphoid cells. Ultimate activation of IL-4 expression takes place through contributions from ubiquitous and induced transcription factors that converge upon the GATA-3-modified locus. Chromatin-modifying complexes required to alter histones and reposition nucleosomes move rapidly to the nucleus after TCR stimulation (Zhao et al., 1998). In addition to being present as protein, GATA-3 transcription accompanies activation, is further augmented by Stat6-mediated signals, and is attenuated by concomitant Stat1- and/or Stat4-mediated signals (Ouyang et al., 2000), which would be generated during induction of type 1 immunity. Both Stat6 and GATA-3, the latter in a Stat6-independent way, promote transcription of c-Maf, a b-ZIP transcription factor expressed in Th2 cells. c-Maf binds and transactivates the IL-4 promoter, and c-Maf-deficient T cells are highly attenuated for IL-4 expression (Kim et al., 1999). In addition to c-Maf, efficient IL-4 expression requires ubiquitous members of the NF-AT and AP-1 families, most notably, NF-ATc1 and JunB, respectively, although complex interplay between activating and repressing members of these transcription families adds regulatory detail (Hartenstein et al., 2002; Ranger et al., 1998; Rengarajan et al., 2002b; Yoshida et al., 1998). Additional

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proteins, including NIP45, IRF4, and TRAF2, have been implicated in modulating IL-4 expression in T cells through various protein–protein interactions (Hodge et al., 1996; Hu et al., 2002; Lieberson et al., 2001; Rengarajan et al., 2002a). In addition to c-Maf, a second critical target of GATA-3 is GATA-3 itself. Auto-activation of the gene by its protein product, together with its transcriptional repression by Stat1/Stat4-mediated signaling, is likely a fundamental process underpinning the polarization of IL-4 expression in differentiating T cell subsets (Ouyang et al., 2000). Additionally, GATA-3 represses IL-12Rb2 expression. Despite its central role in the process of Th2 differentiation, the transcription of GATA-3 itself remains incompletely understood, but the discovery of alternative promoters and silencers suggests careful regulation (Asnagli et al., 2002; Gregoire and Romeo, 1999; Hwang et al., 2002). Activation of the gene involves interplay between NF-AT and NF-kB transcription factors driven to the nucleus in response to TCR stimulation. Transcription is greatly augmented by Stat6 and GATA-3. However, sufficiently prolonged signaling is likely to account for observations of IL-4/IL-4R-/Stat6-independent Th2 cell differentiation, since once a threshold has been reached, GATA-3 is alone sufficient to program downstream commitment of the locus independent of these signals (Finkelman et al., 2000; Jankovic et al., 2000; Mohrs et al., 2001b). Deletion of mel-18, a polycomb group gene, resulted in failure to sustain GATA-3 induction with subsequent inability to develop Th2 cells (Kimura et al., 2001). Polycomb genes are typically associated with silencing, raising the possibility that mel-18 restricts expression of inhibitory GATA-3interacting proteins, such as FOG-1 and ROG, which impede GATA-3mediated transcription (Fox et al., 1999), or SOCS5, a Th1-associated negative regulator of IL-4Ra/Stat6 signaling (Seki et al., 2002). Further work is needed. III. Stabilization of IL-4 Expression in T Cells

Activation of IL-4 transcription, initially a Stat6-independent process in naive CD4 T cells, is not sufficient to stabilize the locus for committed expression by daughter cells (Grogan et al., 2001). Persistent signals are required, presumably to achieve threshold levels of GATA-3 required to drive the process in a cell autonomous manner. Although sustained TCR and co-stimulatory signals can be sufficient for Th2 development, this threshold is reached much more readily by the addition of IL-4Ra/Stat6-mediated signals (Ouyang et al., 2000). Stat6 mediates induction of growth factor independent-1 (Gfi-1), a transcriptional repressor, which dramatically augments proliferation and survival of cells expressing GATA-3 (Zhu et al., 2002). Requirements for potent, sustained, signaling and/or exogenous IL-4 create thresholds for Th2 development. Using a sensitive knockin GFP reporter,

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analysis after TCR activation suggested that IL-4 transcription is initiated from both alleles in CD4 T cells (Mohrs et al., 2001b). Despite this observation, analysis of differentiated Th2 clones from wild-type or knockin reporter mice have revealed both monoallelic and biallelic expression that is stable over repeated stimulations (Bix and Locksley, 1998; Hu-Li et al., 2001; Riviere et al., 1998). Levels of IL-4 transcribed may also be modulated and reflect variability in the extent of chromatin stabilization across the gene (Guo et al., 2002). Taken together, the results are consistent with a gene that is transcriptionally competent at the time of TCR stimulation, but then variably modulated at individual alleles in response to subsequent signals. The resultant cells likely consist of a spectrum from clones with chromatin modified across the entire IL-4/IL-13/IL-5 cluster to clones modified across both IL-4 alleles to clones modified across a single IL-4 allele. A silencer element has been identified in the 30 untranslated region of the IL-4 gene near areas which may nucleate methylation (Kubo et al., 1997; Lee et al., 2002). Consensus binding sites for the Bcl6 and Runx repressors are within the sequence, but their functional significance remains unknown. The pivotal role for Stat6 is borne out by the severe deficit in type 2 immunity in Stat6-deficient mice. These animals, as well as IL-4Ra-deficient mice, are unable to expel intestinal helminths and fail to mount normal allergen-induced mucosal responses (Urban et al., 1998). These models are biologically complex, however, and may represent the confluence of known or likely roles for Stat6 in induction of tissue-recruiting chemokines (Cuvelier and Patel, 2001), appropriate vascular adhesins (Issekutz et al., 2001), and T cell selectin ligands (Wagers et al., 1998). Thus, some of the Stat6-deficient phenotype may reflect failure to localize the appropriate effector cells rather than inherent failures in Th2 differentiation itself (Mathew et al., 2001). Rechallenge of Stat6-deficient mice with helminths, however, suggests a primary role for Stat6 in stabilized Th2 effector development. Primary infection of Stat6-deficient mice results in a systemic IL-4 response not different from that in challenged wild-type mice. IL-4 production failed to be maintained, however, and, in contrast to wild-type mice that generated an enhanced memory response, rechallenged Stat6-deficient mice reiterated a primary IL-4 response (Finkelman et al., 2000). These results suggest that IL-4 production by innate immune cells is Stat6-independent. Further, since analysis with knockin reporter mice demonstrated normal IL-4 induction in naive T cells in lymph nodes after helminth challenge, the data suggest that IL-4 production by T cells, while initially Stat6-independent, cannot be sustained in the absence of Stat6 (Mohrs et al., 2001b). Whether these effects are direct or mediated by a requirement for tissue localization of Th2 cells for their terminal differentiation will require further study.

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A consistent observation in Th2 cells is the localization of the IL-4 genes to euchromatin domains in the nucleus, in contrast to Th1 cells, which show repositioning of IL-4 alleles in apposition to heterochromatin, consistent with epigenetic silencing (Grogan et al., 2001). Localization to heterochromatin is used to silence other genes in lymphocytes, frequently through interactions with ikaros family proteins that mediate interactions with centromeric domains (Fisher and Merkenschlager, 2002). Ikaros sites are dispersed widely through the type 2 cytokine locus, including 4 sites clustered in the CNS-1 element. CNS-1-deficient Th1 cells fail to reposition the IL-4 genes to heterochromatin, suggesting that the same DNA elements targeted by activating complexes in Th2 cells may be occupied by silencing complexes in Th1 cells that mediate terminal sequestration of the locus (Grogan and Locksley, unpublished studies). The plasticity of IL-4 expression in differentiated Th2 populations remains an area of intense interest. Early studies suggested that repeated stimulation under polarized Th2 conditions drove T cells to irreversible patterns of cytokine expression (Murphy et al., 1996). Whether such conditions occur in vivo is unknown. Studies in both human and mouse, however, have suggested a spectrum of cytokine-producing cells based on their capacity to respond rapidly to antigen recall. In the mouse, where more detail has been developed in studies of CD8 cells, a hierarchical lineage from tissue-occupying effector cells to circulating, effector memory cells to circulating, central memory cells, has been defined (Wherry et al., 2003). However, analysis of human T cell populations has suggested a model of linear differentiation from central memory to effector memory cells (Sallusto and Lanzavecchia, 2001; Sallusto et al., 1999) (although contradictory evidence has been reported in human and mouse systems (Baron et al., 2003; Wu et al., 2002) ). Central memory cells keep their proliferative potential and give rise to effector/memory cells rapidly upon antigen encounter. Emerging data on CD4 T cells, although incomplete, are consistent with a similar lineage scheme (Lohning et al., 2002). By this model, Th2 effector/memory cells are tissue-infiltrating cells poised for rapid, stereotyped, type 2 cytokine secretion, which may have limited proliferative potential. In contrast, Th2 central memory cells retain the capacity for clonal expansion and antigen sampling in lymph nodes. Although commitment to transcribe type 2 cytokines is presumably maintained in each of these populations, the capacity to activate other cytokines, including IFNg, may be greater in central memory cells. Studies using human CD4 T cells have suggested substantial cytokine flexibility among polarized effector and memory Th subsets (Messi et al., 2003). It remains unclear how in vitro-generated Th2 cells compare to cells generated in vivo, or how reliable the use of surface markers for the identification of various subsets in vivo remains, making

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generalizations difficult. These remain crucially important questions for further study in understanding, for instance, how chronic type 2 afflictions such as allergy and asthma are sustained. For instance, whether tissue Th2 effector cells undergo activation-induced cell death, as posited for Th1 tissue effectors (Hayashi et al., 2002), remains unknown. IV. Phenotype and Genotype Analysis of Th2 Cells

Polarization of Th subsets in vitro is associated with modulation of surface receptors that provide information about the cytokine milieu. In vitro, differentiating Th2 cells lose IL-12Rb2 and IL-18 receptor surface expression induced at the time of activation, while IL-4R signaling remains robust, perhaps reflecting failure to upregulate SOCS5, as occurs during Th1 differentiation. The IL-1R family member, T1/ST2 (IL-1RL1), is induced on the surface of Th2 cells, and can function as a co-stimulatory signal for proliferation and type 2 cytokine production (Meisel et al., 2001). CRTH2 is a 7-transmembrane, G-protein coupled receptor related to N-formyl peptide receptors that is induced on Th2 cells and facilitates chemotaxis to prostaglandin D2, the major prostaglandin metabolite of activated mast cells (Hirai et al., 2001). The histamine receptor type 1, H1R, which costimulates IFNg production in Th1 cells, is down-regulated on Th2 cells (Jutel et al., 2001). Distinct surface adhesins, including a4b7 integrin, the ligand for mucosal MAdCAM-1, are induced, whereas upregulation of the fucosyltransferase, Fuc-TVII, required for the synthesis of P- and E-selectin ligands, is dependent upon IL-12/Stat4 and, hence, not typically found on Th2 cells. Patterns of Th2 chemokine receptors, while complex and combinatorial, have demonstrated some bias towards recognition by mucosal and allergen-generated chemokines (Luther and Cyster, 2001). While more study is needed, the collective data suggest that Th2 cells acquire the potential to enter mucosal or epithelial sites in response to chemokines commonly associated with allergic inflammation. Several groups have used microarrays to compare developing or stable Th1 and Th2 cell lines in efforts to identify subset-specific transcripts. Transcripts with a bias towards expression in Th2 cells include cytokines (IL-4, IL-13, IL-5, IL-24), transcription factors (GATA-3, Stat6), receptors (T1/ST2), and adhesins (integrin b7). These trends have been seen in some, but not all, studies, perhaps reflecting varied differentiation methods and different arrays (Chtanova et al., 2001; Nagai et al., 2001; Rogge et al., 2000). Studies of Th2 cells purified from distinct tissue compartments in vivo are lacking, and will be informative in understanding the function of Th2 cells as they traffic to sites of infection.

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V. Mutations Impacting IL-4 Expression in T Cells

Many mutations affecting IL-4 expression from Th2 cells, including deficiencies in IL-4, Stat6, IL-4Ra, GATA-3 (via anti-sense or dominant negative strategies) and c-Maf, or targeted deletions of cis-acting regulatory elements, have already been noted. A number of other gene deletions have effects on Th2 cell development in vitro or in vivo. In general, these can be divided into deletions or additions of exogenous soluble or surface ligands, deletions in signaling pathways, deletions in transcription factors, or imbalances resulting from deletions of key elements required for type 1 immunity. Additional uncharacterized mutations impact pathways likely related to type 2 immunity, such as serum IgE. Examples are listed in Table I, although we focus here on relatively recent observations and do not provide an exhaustive overview. Effects of deficiencies in CD4, lck, itk, LAT (linker for activation of T cells), Vav, PKCu and SLP76, however, begin to outline a linear signaling pathway, that, when compromised, impairs activation of IL-4 gene expression (Aguado et al., 2002; Fowell et al., 1997, 1999; Hehner et al., 2000; Yamashita et al., 1998). Small molecule inhibitors targeting aspects of this pathway might be rationally explored for their capacity to impede Th2 differentiation by pharmacologic means.

VI. Expression of IL-4 in Non-Th2 Cells

IL-4 expression has been documented from CD8 T cells, NK T cells, gd T cells, mast cells, basophils, and eosinophils. Other cell types, such as B cells and dendritic cells, have been reported to produce IL-4 under defined circumstances, but such observations remain incompletely examined. Studies of IL-4 expression in non-Th2 cells should be informative in exposing core genetic programs required for hardwiring and expressing genes from the type 2 cytokine locus. Still lacking is definitive information regarding the requirements for type 2 cytokines generated by innate cells for the terminal differentiation and/or tissue localization of effector Th2 cells. A. Tc2 Cells The name ‘‘Tc2’’ was introduced by Mosmann’s group, who showed that mouse CD8 T cells could be differentiated into stable type 2 cytokineproducing clones in vitro after stimulation with alloantigen and IL-4 (Sad et al., 1995). Evidence for the existence of these cells in vivo came from studies with AIDS patients, who showed relatively high numbers of Tc2 cells in blood and skin (Romagnani et al., 1994). More recent reports show that Tc2 cells are increased in the elderly, and that high numbers of Tc2 cells correlated with a better humoral immune response after influenza vaccination (Schwaiger et al., 2003; Yen et al., 2000). Tc2 cells can provide B cell help by secretion of

TABLE I Mutations Impacting Type 2 Immunity Mutation

Phenotype

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Ligand/receptor interactions ICOS/ Reduced IL-2, IL-4, IL-5, IL-13 Increased EAE susceptibility OX40/ Reduced IL-4, IL-5, IgE, decreased eosinophilia OX40L/ Reduced IL-4, IgE, impaired worm expulsion TNF/ Reduced Th2 cytokines DR6/ Increased Th2 cytokines

Model In vitro, asthma, immunization Asthma H. polygyrus Asthma Immunization, in vitro Aspergillus, asthma In vitro, asthma

Reference(s) (Dong et al., 2001)

C3aR/ CD81/

Reduced eosinophilia, reduced IL-4, IL-5, IL-13 Decreased Th2 cytokines, normal serum IgE

Tim-1 polymorphisms TSLP administration

Locus involved in airway hyperreactivity Produced by epithelial cells, favors Th2 differentiation, increased expression in atopic dermatitis Increased mastocytosis, dermatitis, Th2 cytokines in skin

Asthma Human, in vitro

(Jember et al., 2001) (Ekkens et al., 2003) (Matheson et al., 2002) (Liu et al., 2001a; Zhao et al., 2001) (Drouin et al., 2002) (Deng et al., 2000; Deng et al., 2002) (McIntire et al., 2001) (Soumelis et al., 2002)

Spontaneous

(Konishi et al., 2002)

Increased IgE Impaired IgG1 production Increased Th2 cytokines, IgE, eosinophilia

Spontaneous

(Ozaki et al., 2002)

In vivo administration Immunization

(Fort et al., 2001)

IL-18 transgene, skin-specific IL-21R/ IL-21R/IL-4/ IL-25 administration IL-27/

Decreased IL-4 from NK T cells; conventional T cells normal

(Nieuwenhuis et al., 2002)

Signaling molecules LAT Y136F point mutation Vav dominant negative Itk/ Fyn/

Impaired T cell development, increased IL-4, lymphoproliferation and eosinophilia Decreased IL-4 transcription Impaired IL-4 expression and NF-ATc nuclear translocation Increased Th2 cytokines, eosinophilia

Spontaneous

In vitro In vitro, asthma LCMV, L. major

P85 PI3K/

Decreased proliferation and Th2 cytokines; normal survival Increased Th2 cytokines, IgE, eosinophilia Decreased Th2 cytokines, IgE, Increased CD8 and Th1 response Defective B cell memory Mutated in XLP Reduced intestinal mast cells, delayed worm expulsion

Gab2/

Decreased mast cell function, impaired FceRI signaling

IRS2/ Tyk2/ SAP/

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Transcription factors STAT6/

Mbd2/ Mel-18/

Normal initial IL-4 response Impaired Th2 tissue localization and IL-4 memory response Increased IL-4 expression Decreased Th2 cytokines, impaired worm expulsion

NFATc2/NFATc3/ NFATp/ IRF4/ IRF4 over-expression

IL-4-independent Th2 differentiation Reduced early IL-4 production Decreased Th2 cytokines Increased Th2 cytokines

In vitro In vitro, L. major In vitro, asthma

Human Strongyloides venezuelensis in vitro, immunization

(Aguado et al., 2002; Sommers et al., 2002) (Hehner et al., 2000) (Fowell et al., 1999) (Kudlacz et al., 2001; Tamura et al., 2001) (Wurster et al., 2002) (Seto et al., 2003) (Czar et al., 2001; Wu et al., 2001) (Crotty et al., 2003) (Morra et al., 2001) (Fukao et al., 2002) (Gu et al., 2001)

N. brasiliensis

(Finkelman et al., 2000) (Mohrs et al., 2001b)

In vitro In vitro, N. brasiliensis In vitro Immunization In vitro In vitro

(Hutchins et al., 2002) (Kimura et al., 2001) (Rengarajan et al., 2002b) (Hodge et al., 1996) (Rengarajan et al., 2002a) (Hu et al., 2002) (continues)

TABLE I (continued) Mutation Foxp3

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Miscellaneous Itch/ SPINK5 CC10/

Phenotype

Model

Reference(s)

Mutated in XLAAD (allergy/autoimmunity)

Human

Mutated in Scurfy mouse (lymphoproliferation) Essential for regulatory T cell (Treg) development

Spontaneous In vitro

(Bennett et al., 2001 Chatila et al., 2000) (Brunkow et al., 2001) (Hori et al., 2003)

E3 ubiquitin ligase: enhanced Th2 cytokines, increased serum IgE, IgG1 Serine protease inhibitor: mutated in Netherton syndrome (atopy/allergy) Surfactant cell secreted protein: increased Th2 cytokines, eosinophilia

In vitro

(Fang et al., 2002)

Human

(Chavanas et al., 2000; Walley et al., 2001) (Chen et al., 2001)

Asthma

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IL-4 (Maggi et al., 1994), but, like Tc1 cells, display cytotoxic potential (Sad et al., 1995). Further, it has been shown that Tc2 cells could reduce the growth of established lung metastases in a mouse tumor model (Dobrzanski et al., 1999). Conditions that lead to the differentiation of Tc2 cells in vivo and their true physiological role remain incompletely characterized. B. NK T Cells NK T cells are a relatively small population of T cells that express the NK cell marker, NK1.1, and a restricted repertoire of ab T cell receptors (Kronenberg and Gapin, 2002). The most prevalent NK T cells fortuitously recognize the nonclassical MHC molecule, CD1d, when loaded with agalactosylceramide (a-GalCer), which presumably mimics the unknown selecting ligand. These cells, designated invariant NK T cells (NK Ti) because of their use of the same TCRs in mouse and human, arise from a common T cell precursor after stochastic rearrangements generate a canonical receptor that permits selection on CD1d presented by double-negative thymocytes. In contrast to ab T cells, NK T cells acquire the capacity for rapid IL-4 and IFNg production during thymic development (Benlagha et al., 2002). After migration to liver, bone marrow, and spleen, NK Ti cells undergo terminal differentiation. Essentially all of the early IL-4 that appears in blood after administration of anti-CD3 or a-GalCer is from NK Ti cells. Evidence that these cells are required for type 2 immunity is lacking, however. Their rapid cytokine responses have been exploited to bias immune responses in various infectious diseases, autoimmune, and tumor models. Production of IL-4 by NK Ti cells is unaffected by Stat6-deficiency, attenuated in itk-deficiency, and, unexpectedly, highly compromised in the absence of EBI3, an IL-12 p40 homolog that dimerizes with IL-12 p35 or its homolog, p28, to create IL-23. Despite data demonstrating induction of IFNg by IL-23 in T cells, NK Ti development and IL-4 production are severely attenuated in the absence of EBI3 (Nieuwenhuis et al., 2002). C. gd T Cells gd T cells constitute 1 to 5% of lymphocytes in the blood of adult animals, but can compose up to 50% of T cells in epithelial tissues such as skin and the gastrointestinal tract. They are not restricted by classical MHC, can recognize soluble protein and nonprotein antigens, and many have cytotoxic function (Carding and Egan, 2002). gd T cells predominate during early fetal development and the expression of IL-4 by fetal thymocytes was first described 15 years ago (Tentori et al., 1988). Human pro-T cells isolated from neonatal thymus also produce IL-4 (Barcena et al., 1991). In vitro cultures of these cells in the presence of IL-4 gave rise to gd T cells, suggesting that development of thymic gd T cells might be regulated by autocrine or paracrine stimulatory

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signals provided by IL-4. Some gd T cells isolated in adult life express the NK1.1 marker and secrete IL-4 upon in vitro stimulation (Azuara et al., 1997). This subset of IL-4 producing gd T cells exists predominantly in the liver and spleen (Gerber et al., 1999). In the mouse, gd T cells can be induced to secrete type 1 or type 2 cytokines after polarizing infections (Carding et al., 1993; Ferrick et al., 1995). Further, in a pulmonary allergic asthma model, gd T celldeficient mice had reduced recruitment of effector cells into the lung and attenuated IgE levels. Administration of recombinant IL-4 during immunization of gd T cell-deficient mice restored these responses, suggesting that gd T cells might contribute to early IL-4 production during the development of allergic asthma (Zuany-Amorim et al., 1998). D. Mast Cells and Basophils Mast cells and basophils express the high-affinity IgE receptor and release inflammatory mediators and cytokines after IgE cross-linking (Kinet, 1999; Williams and Galli, 2000). Both cell types express IL-4 (Bradding et al., 1992; Brown et al., 1987; MacGlashan et al., 1994; Plaut et al., 1989; Seder et al., 1991). Further, mouse and human basophils dramatically upregulate IL-4 transcription and release IL-4 protein within one hour of allergen challenge (Genovese et al., 2003; Luccioli et al., 2002). This effect was dependent on the high-affinity IgE receptor and was also observed in KitW/KitWv mice, which are genetically mast cell-deficient, suggesting that basophils might be an important source for early IL-4 in vivo. IL-4 expression in mast cells is Stat6- and GATA3-independent, in contrast to Th2 cells, but requires Stat5 and GATA-1/-2 (Sherman, 2001; Weiss and Brown, 2001). E. Eosinophils Distinct from other IL-4-expressing cells, human eosinophils store preformed IL-4 in granules, which can be released rapidly upon stimulation (Bandeira-Melo et al., 2001; Moqbel et al., 1995). In vitro, mouse eosinophils can secrete IL-4 within 18 hours after stimulation, which is blocked by actinomycin D. Thus, mouse eosinophils likely do not store preformed IL-4 but rapidly synthesize it after activation (Justice et al., 2002). In Schistosoma egg granulomas, eosinophils appear to be the major source of IL-4 (Rumbley et al., 1999). Tissue-infiltrating eosinophils were also demonstrated to express IL-4 after helminth infection, even in the absence of an adaptive immune response (Shinkai et al., 2002). F. B Cells A 2000 study has shown that B cells can be differentiated into cytokinesecreting effector B cells (Harris et al., 2000). Like T cells, B cells could be polarized to secrete type 1 or type 2 cytokines. Whether they play an important role in vivo remains to be determined.

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VII. Where Does Type 2 Immunity Operate?

In vivo, type 2 immunity is most readily apparent after mucosal challenge with helminths and allergens, and, in each, depletion of Th2 cells ablates or substantially attenuates the response. Systemically, type 2 responses can function in a regulatory role by abrogating potential inflammatory damage mediated by activated type 1 immune cells (Matsukawa et al., 2001). The propensity for type 2 immune responses to develop at epithelial barriers suggests several hypotheses regarding this apparent geographic predeliction, none of which are mutually exclusive (Fig. 2a). First, antigens acquired across epithelial barriers might possess intrinsic properties that drive type 2 immune responses. Thus, many epithelial antigens, whether derived from exogenous allergens, invading helminths, or biting insects, share the properties of stability to harsh conditions and intrinsic proteolytic activity. Such biophysical attributes may enhance transepithelial delivery to sentinel dendritic cells or contribute to modulating signals via unknown molecular recognition receptors. Crude cocktails of helminthderived antigens frequently contain type 2 polarizing activities (Holland et al., 2000). Despite the explosion of information regarding the role of Toll family proteins as molecular recognition sensors important in mediating type 1 immune responses (Medzhitov, 2001), definitive identification of a pattern recognition receptor critical for initiating type 2 immunity remains elusive (Fig. 2b). A second possibility is that distinct types of dendritic cells populate epithelial and mucosal surfaces. The ability to identify different types of DCs with differing capacity to activate naive T cells to develop into Th1 or Th2 cells has introduced further complexity (Liu et al., 2001b; Patterson, 2000; Reis e Sousa, 2001). Differential recruitment of dendritic cells from the periphery or blood, such as ‘‘pre-DC-2’’ plasmacytoid DCs, may impact T cell polarization through varied display of surface ligands that can affect cytokine production (Table I). IL-4 itself, by regulating expression of sampling receptors, such as DC-SIGN, can affect antigen delivery to the draining lymph node (Relloso et al., 2002). Delivery of antigens to mucosal sites can bias toward type 2 immune responses (Constant et al., 2000). Much more information is needed regarding which populations of DCs become activated during type 2 immunity. A third hypothesis, alluded to earlier, remains the possibility that terminal Th2 differentiation in vivo occurs in tissue, and that signals that mediate this occur predominantly in epithelia. Accumulating evidence supports the speculation that naive CD4 T cells might be ‘‘poised’’ after undergoing clonal expansion in the draining lymph node, but terminally differentiated by cytokine signals, like IL-4, delivered later, perhaps after tissue infiltration (Doyle et al., 2001; Mohrs et al., 2003; Wang and Mosmann, 2001). The positioning and/or recruitment of IL-4-expressing innate cells, including mast cells, eosinophils,

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Fig 2 Initiation and expression of type 2 immunity. (a) A schematic diagram depicting three levels at which Type 2 immune responses may be regulated. (b) A simplified comparison of how type 1 and type 2 responses are initiated. Recognition of pathogen-associated molecular patterns (PAMPS) by pattern recognition receptors (PRRs) results in the production of cytokines which modulate T cell differentiation in the draining lymph nodes. While the basic elements underlying all of these events have been elucidated for Type 1 immunity, very little is known about how recognition of type 2 pathogens is linked to Th2 differentiation.

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and basophils, to invaded barriers would then serve to deliver terminal differentiation signals to poised T cells entering the tissue. In turn, developed Th2 cells reinforce the growth, recruitment, and activation of mast cells, basophils, and eosinophils, resulting in sustained, autoreinforcing, changes at epithelial barriers. The latter, although incompletely characterized, include the development of changes in water and ion exchange, mucus hypersecretion, and, ultimately, subepithelial fibrosis, that together impede access to blood by arthropods or helminths, or tissue invasion by widely prevalent environmental allergens (Madden et al., 2002). Unregulated, however, epithelial type 2 responses may contribute to the clinical manifestations of allergy and asthma. The overlapping phenotype of type 2-associated pathology generated by overexpression of any of the genetically linked type 2 cytokines—IL-4, IL-13, IL-5, and IL-9 (on the same chromosome in humans but the latter split off in the mouse)—suggests integrated functional redundancies (Lee et al., 1997; Rankin et al., 1996; Temann et al., 2002; Zhu et al., 1999). Pairwise comparisons of variable combinations of knockouts established that IL-4 alone, even in the absence of IL-13, IL-5, and IL-9, could mediate all of the attributes of type 2 immunopathology, given a sufficiently strong stimulus in vivo (Fallon et al., 2002). Thus, type 2 immunity consists of an orchestrated response underpinned by innate cells but centrally mediated, in its adaptive phase, by Th2 cells. VIII. Concluding Remarks

We have attempted to summarize recent insights regarding the generation of Th2 cells and factors regulating expression of IL-4, the canonical cytokine secreted by these cells. Th2 cells orchestrate a stereotyped cell response characterized by eosinophil and basophil infiltration, mast cell hyperplasia, and changes in epithelial mucus and water secretion. Th2 cells have been demonstrated to mediate protection against a variety of intestinal helminths but also ectoparasites that invade at epithelial barriers. When activated systemically, Th2 cells protect against the tissue-damaging proinflammatory actions of Th1 cells. When dysregulated, Th2 cells drive chronic atopic diseases of wide prevalence, such as allergy and asthma. Basic insights into the understanding of the generation, activation, tissue localization, and turnover of Th2 cells will provide numerous opportunities for sustained benefits to human health. Acknowledgments The authors regret being unable to cite all of the relevant original literature due to space constraints. Supported in part by the Howard Hughes Medical Institute and grants from the National Institutes of Health (AI26918, AI30663, HL56385). RML is a Larry Ellison Global Infectious Diseases Senior Scholar.

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