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Transcriptional regulation of Th1/Th2 polarization Jyothi Rengarajan, Susanne J. Szabo and Laurie H. Glimcher The two polarized T helper (Th)

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critical aspect of the immune DCs induce IL-4 production8,9. Overall, the by their signature cytokines, defense against infection mutually antagonistic effects of IL-4 and interferon g (IFN-g) and interleukin by pathogens is mediated IFN-g regulate Th1/Th2 balance and subseby the helper function of quent polarization. What is the molecular 4 (IL-4) respectively. Understanding 1 1 CD4 T cells. When CD4 T helper (Th) cells basis for this polarization? Investigating the the transcriptional regulation of encounter microbial antigens presented by transcriptional regulation of IL-4 as the hallantigen presenting cells, they are activated mark cytokine for Th2-type, and IFN-g for cytokine expression is therefore and differentiate into two functionally disTh1-type differentiation, and by determining critical for elucidating the process of tinct subsets (Fig. 1). Th1 cells secrete the the DNA elements and proteins involved in Th cell differentiation. Ubiquitous cytokines interleukin 2 (IL-2), interferon g the transcription of these genes, has proven (IFN-g) and tumor necrosis factor b (TNF-b), to be a rewarding approach. and tissue-specific transcription and are efficient in eliminating intracellular factors, as well as chromatin pathogens like Leishmania via macrophage remodeling of genomic loci have Transcriptional regulation of activation. Th2 cells secrete the cytokines cytokine gene expression IL-4, IL-5, IL-10 and IL-13, which effect hubeen implicated in IL-4 and IFN-g Signals through the TCR as well as through moral immunity to helminthic parasites and regulation. We propose a model of cytokine receptors elicit a complex series of are responsible for immune responses to Th1/Th2 polarization based on the molecular interactions that culminate in the persistent antigens, for instance allergens1–3. The two helper subsets also cross-regulate transcription of cytokine genes. These signal balance between Th1- and Th2each other, so the balance between Th1 and transduction cascades have emerged as imspecific transcription factors. Th2 cytokines can determine whether the portant regulators of Th differentiation but immune response is appropriate or will terare beyond the scope of this article (reviewed minate in detrimental immunopathologies. in Ref. 10). Faithful transcription of genes is Overproduction of Th1 cytokines has been implicated in delayed- orchestrated by the cooperative interactions of multiple ubiquitous type hypersensitivity reactions and autoimmune diseases. Th2 cyto- and cell-type-specific transcription factors, bound to multiple regukines recruit eosinophils and activate mast cells; thus dysregulation latory elements in the promoters of genes. These protein–protein and of Th2 cytokines can lead to allergic and inflammatory conditions3. protein–DNA contacts, together with their interactions with the basal transcriptional machinery, result in combinatorial regulation of genes, which confers specificity and precision to the transcription Cytokines regulate Th cell differentiation process11,12. The stringent regulation of cytokine genes during Th The polarized subsets Th1 and Th2 both develop from the same Th differentiation has made them an excellent model for studying precursor (Thp) and differentiate into the two phenotypes via a com- stage- and tissue-specific transcriptional regulation. plex developmental process. The dose of antigen, strength of signal through the T-cell receptor (TCR) and costimulation all influence the initiation of Th differentiation2,4. An important insight was obtained Ubiquitous and Th2-specific factors mediate IL-4 from the observations that the antigen-activated naive Thp cell can expression be induced to differentiate into the Th1 or Th2 lineage in vitro by the IL-4 production is tightly regulated and the cytokine is expressed in addition of exogenous cytokines5. IL-12 promotes IFN-g production a subset of immune cells namely Th2 cells, mast cells, NK T cells, and Th1 development via signaling pathways that lead to activation basophils, eosinophils and gd T cells. The quest to delineate the molof Stat4 (signal transducer and activator of transcription 4) (Ref. 6). ecular basis for cell-specific and activation-dependent IL-4 expression Ligation of the IL-4 receptor by IL-4, leading to Stat6 activation, can led several groups to investigate the promoter region of the IL-4 drive a naive Th cell down a Th2 differentiation pathway6. Thus, gene (Reviewed in Refs 13–15). It was shown that 87 bp of sequence cytokines have emerged as critical inducers of Th subset develop- upstream of the transcription initiation site was sufficient for TCRment (Fig. 1). Specific dendritic cell (DC) subsets located in distinct inducible and Th2-specific expression, and five sites (P0–P4), located microenvironments in the spleen appear to bias Th subset develop- within the 59 flanking region were shown to be critical for inducible ment by eliciting critical cytokines7. CD8a1 DCs produce IL-12 in expression of IL-4 (Ref. 15) (Fig. 2a). Proteins of the nuclear factor of response to microbes and induce IFN-g and IL-2, whereas CD8a2 activated T cells (NFAT) family bind specifically to these sites and PII: S0167-5699(00)01712-6

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IL-12

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IL-12R CD8α– DC Th2 IL-4 IL-12Rβ2

IL-4 IL-4 IL-5 IL-10 IL-13 • Antibodies • Mast-cell degranulation • Eosinophil activation Immunology Today

Fig. 1. Overview of Th cell differentiation. A naive CD41 T cell is activated via the TCR when it encounters antigen (for example, derived from a microbe or allergen) presented by an antigen presenting cell. Once activated, the Th cell starts to proliferate and secrete IL-2, and expresses the IL-12 receptor (IL-12R). On encountering IL-12 secreted by macrophages and/or on contact with CD8a1 dendritic cells (DCs), a Th1 differentiation program is initiated. The IL-12Rb2 chain is upregulated in the developing Th1 cell and ligation of the IL-12R by IL-12 leads to the activation of Stat4 and initiation of the Th1 differentiation program. IL-4 produced intrinsically by the Thp and extrinsically by several cell types as well as contact with CD8a2 DCs induces differentiation into the Th2 subset and downregulation of IL-12Rb2 expression. Ligation of the IL-4R (expressed on the naive CD41 T-cell surface) by IL-4 activates Stat6 and initiates the Th2 differentiation program. Abbreviations: TCR, T cell receptor; IL-4, interleukin 4; Stat6, signal transducer and activator of transcription 6; Stat4, signal transducer and activator of transcription 4; Th1, T helper 1; Thp, T helper precursor.

cooperate with activator protein (AP-1) factors like Fra and Jun at sites P1 and P4 to induce IL-4 transcription. This activation of NFAT proteins is inhibited by the immunosuppressants cyclosporin A (CsA) and FK506, and is reflected by the CsA-sensitivity of the IL-4 proximal promoter (reviewed in Refs 13, 14). The in vivo role of NFAT factors in IL-4 expression, however, points to greater complexity. Whereas mice that lack NFATc1 (NFATc) have impaired IL-4 production, mice that lack two of the three known lymphoid NFAT family members, NFATc2 and NFATc3, (NFATp and NFAT4) displayed extreme overproduction of IL-4 and other Th2 cytokines, revealing a negative regulatory role for NFAT in IL-4 production16,17. Whether this is directly at the level of transcription or via the regulation of target genes that negatively regulate cytokine signal transduction is presently unknown. What is clear is that the balance between NFAT family members is critical for Th1/Th2 polarization.

c-Maf and IL-4 expression in Th2 cells Although NFAT and AP-1 proteins are important for IL-4 transcription, they are present in both Th1 and Th2 subsets and, thus, do not explain the Th2-restricted expression of IL-4. A major advance

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towards elucidating this came with the confirmation that the proto-oncogene c-Maf is specifically expressed in Th2 but not Th1 cells. c-Maf, a basic leucine zipper transcription factor, was shown to bind to a maf response element (MARE) within the IL-4 proximal promoter18. c-Maf strongly transactivates an IL-4 promoter reporter construct in vitro and, in synergy with NFATc2 and NIP45 (NFAT interacting protein 45), can elicit endogenous IL-4 production in M12 B lymphoma lines19. Mice deficient in c-Maf have impaired IL-4 production but produce no changes in the other Th2 cytokines like IL-5, IL-10 and IL-13 (Ref. 20). Although mice overexpressing c-Maf show preferential Th2 responses, Th1 cells derived from these mice do not transcribe IL-4 (Ref. 21). Thus, cMaf is specific for IL-4 and is critical for high levels of IL-4 production but is not sufficient for the initiation of IL-4 transcription.

GATA-3 and Th2 differentiation

The search for additional genes that are preferentially expressed in Th2 cells led to the isolation of GATA3, a zinc finger protein that was originally identified as binding the TCRa gene enhancer via a WGATAR sequence. GATA3 is expressed in the course of Th2 differentiation through pathways that probably involve the IL-4 dependent activation of Stat6, but is undetectable in Th1 cells22–24. Although Stat6 activation is required for Th2 differentiation, as shown by the inability of Stat6-deficient T cells to differentiate into the Th2 phenotype, its direct role in IL-4 transcription remains unclear6. By contrast, consensus and non-consensus GATA3 elements have been identified in the IL-4 locus that significantly increase transactivation of the IL-4 promoter by GATA3 (Ref. 25). In addition, GATA3 has been shown to directly regulate IL-5 and IL-13 expression and thus appears to play a more global role in regulating Th2 cytokines26,27. In one study, low levels of IL-4 and IL-5 were also detectable in committed Th1 cells when transduced with GATA3 and stimulated with cAMP28. Interestingly, GATA3 has also been shown to inhibit the production of IFN-g. Retroviral transduction of GATA3 into developing Th1 cells resulted in a decrease in IFN-g production and subsequent Th1 development, possibly by repressing the expression of the IL12Rb2 chain and the responsiveness of these cells to IL-12 (Ref. 24). The ability of GATA3 to promote Th2 lineage commitment with concomitant inhibition of Th1 development suggests a key role for this transcription factor in determining a Th1 or Th2 fate. With this information, picture the following scenario (Fig. 1): (1) encountering antigen in a microenvironment that favors Th2

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differentiation activates a naive Thp cell; (2) signals delivered through the TCR and the provision of IL-4 from non-T cells as well as from the Th cell itself leads to Stat6 activation; (3) GATA3 expression is induced and promotes Th2 development, represses IL12Rb2 and, thus, inhibits IFN-g production; and finally (4) TCR-induced c-Maf expression facilitates further increases in IL-4 transcription in cooperation with NFAT proteins and NIP45 leading to Th2 polarization.

Long range regulatory elements and chromatin structure in IL-4 expression

(a) IL-4 regulatory regions NIP45 c-Maf TATA box +1 NFAT NFAT

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In addition to the cooperative assembly of (b) IFN-γ regulatory regions ATF-2 ATF-2 multiple known and as yet unknown factors GATA fos jun and cofactors necessary for IL-4 expression, AP-2 NF-κB AP-1 fos jun STAT YY1 recent evidence for regulated changes in E-site –796 –761 –266 –186 –98 –70 –44 chromatin structure and the role of distant control regions (for example, enhancers and HS sites locus control regions) during Th differentiaI II III tion has added a further dimension to this 1 kb model of Th2 polarization (reviewed in Refs 13, 29). Researchers were prompted to examexon 1 STAT 2 3 4 ine the remodeling of the chromatin archiNF-κB Immunology Today tecture at cytokine loci in light of the observation that polarized effector cells exhibit higher levels of cytokine expression and Fig. 2. (a) IL-4 regulatory regions: the IL-4 proximal promoter with cis and trans elements and map more rapid kinetics of activation compared of IL-4 locus showing DNAse I HSs (b) IFN-g regulatory region and map of locus with DNAse I HS with naive cells. In addition, transgenic mice sites (Adapted from Ref. 32). Abbreviations: IL-4, interleukin 4; DNAse I HS, DNAse I hypersensitivity expressing varying lengths of the proximal site; IFN-g, interferon g. IL-4 promoter failed to recapitulate endogenous levels of IL-4 expression, suggesting additional elements identified 39 of the IL-4 gene that appears to bind GATA3 (Ref. 35). necessary for optimal expression30,31. Furthermore, the genes encod- NFATc2 was also shown to specifically bind to this site in stimulated ing the Th2 cytokines IL-4, IL-5 and IL-13 are clustered in both Th2 cells but not in Th1 cells35. Putative regulatory sequences involved in controlling the IL-4/ILthe mouse and human genomes, suggesting coordinate regulation of 5/IL-13 locus have been identified by comparing mouse and human these genes. DNAse I hypersensitivity sites (HSs), which reflect local changes sequences and searching for highly conserved regions36. One such in gene accessibility, were identified in the IL-4 locus during Th2 but element within the locus, conserved non-coding sequence 1 (CNS-1), not Th1 differentiation within 48 hours of naive Th cell activation32 when deleted in the context of cytokine transgenes in mice, led to de(Fig. 2a). The same changes in chromatin were mirrored when GATA3 creased production of cytokines encoded by the transgenes IL-4, IL-5 was introduced into Stat6-deficient cells indicating a role for GATA3 and IL-13. Interestingly, CNS-1 influenced the frequency of Th2 in IL-4 locus remodeling, downstream of Stat6 (Ref. 33). Demethyla- cytokine-producing cells and not the levels of cytokines produced per tion of DNA often parallels remodeling of chromatin leading to gene cell suggesting that CNS-1 may conceivably be involved in activating activation. Decreased methylation at the IL-4 locus was observed the IL-4/IL-5/IL-13 locus by modulating chromatin structure 36. during Th2 but not Th1 development34. It has been proposed that Th2-specific proteins like c-Maf and GATA3 may facilitate IL-4 locus accessibility to TCR inducible factors like NFATs by directly remod- Regulation of IFN-g and commitment to Th1 eling chromatin or indirectly by recruiting modifying enzymes32. differentiation Thus, Th1 cells that lack GATA3 and c-Maf maintain a chromatin Complementary to chromatin changes at the IL-4 locus during Th2 structure non-permissive to IL-4 transcription. Consistent with this differentiation, specific changes occur at the IFN-g locus in the hypothesis, an inducible, CsA-sensitive enhancer has been recently course of Th1 commitment. DNAse I HSs have been observed in the

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Fig. 3. Striking a balance: GATA3 and T-bet – model for Th1/Th2 polarization. Signals through the TCR and cytokine receptors can lead to the initiation of a Th1 program (via Stat4 activation) and the induction of T-bet, which promotes Th1 lineage commitment. Signals that favor the activation of Stat6 induce GATA3 leading to Th2 differentiation. c-Maf is then upregulated leading to increased IL-4 production and Th2 polarization. The middle panel shows the results of Northern blotting of developing Th cells. Naive Th precursors (Thp cells) were purified and differentiated along a Th1 (1 in figure) or Th2 (2 in figure) pathway. mRNA was isolated on days 0, 1, 2, 3 and 5 for Northern analyses. The mutually exclusive expression pattern of T-bet and GATA3 may reflect the ability of these factors to antagonize each other. The relative predominance of T-bet and GATA3 may thus determine Th1/Th2 polarization. first and third introns of the IFN-g gene32 (Fig. 2b). In addition, the locus is hypomethylated in Th1 but not Th2 cells32,37. Although 8.6 kb of genomic sequence can direct T cell-specific expression of IFN-g, the IFN-g promoter has not yet been well characterized and the Th1-specific cis-elements within it have not been identified (reviewed in Refs 13, 29). Two essential regulatory elements have been delineated between 2108 and 240 bp of the IFN-g promoter that mirrors the activation-inducible and CsA- sensitive nature of the endogenous IFN-g gene38. In addition, NFAT sites have been identified

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outside these proximal elements, and members of the NFkB and NFAT protein families appear to bind to specific cis-elements regions within the first intron of IFN-g (Fig. 2b) (reviewed in Ref. 13). The transcription factors IRF-1 and Stat4 have been implicated in Th1 differentiation because mice deficient for each of these factors lack Th1 cells, but the exact roles these factors play in Th1 cytokine generation is unclear6. Thus, although these proteins are clearly important in the transcriptional regulation of the IFN-g gene, none has shed light on the Th1restricted expression of IFN-g.

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A substantial advance in elucidating Th1 lineage commitment and IFN-g expression has recently been provided with the isolation of a novel protein T-bet (T-box expressed in T cells)39. T-bet is a member of the T-box family of transcription factors that regulate several developmental processes. T-bet expression strongly correlates with IFN-g expression; it is specifically upregulated in primary Th cells differentiated along the Th1 but not the Th2 pathway39. Ectopic expression of T-bet in vitro leads to strong transactivation of the IFN-g gene reporter construct. Retroviral transduction of T-bet into primary, developing Th cells induces high levels of IFN-g production. Significantly, transduction of T-bet into fully polarized Th2 cells converts them into IFN-g -secreting Th1 cells and simultaneously represses the Th2 cytokines IL-4 and IL-5 (Ref. 39). T-bet thus appears to function by initiating a Th1 differentiation program while repressing Th2 differentiation.

Striking a balance: T-bet and GATA3

We propose a model for Th1/Th2 polarization that involves a balance between the Th1-specific T-bet and the Th2-specific GATA3 (Fig. 3). The naive Thp cell receives signals through the TCR and costimulatory molecules. If the conditions at the time of activation favor the initiation of Th1 differentiation, for example, IL-12-induced signaling via Stat4, T-bet is induced and promotes Th1 lineage commitment. However, if GATA3 is induced via Stat6 activation, the Th cell is driven down a Th2 pathway followed by upregulation of c-Maf and subsequent Th2 polarization. Factors like NFAT are present in both subsets and inducibly bind to specific sites. The mutually exclusive

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expression pattern of T-bet and GATA3 during Th1 and Th2 differentiation respectively, lends support to this model. Furthermore, the two transcription factors may antagonize the development of the opposite subset by directly regulating each other’s expression as well as by remodeling cytokine loci. Thus, the relative predominance of T-bet and GATA3 may determine the Th1/Th2 fate of a Thp cell. Uncovering the precise signals that induce GATA3 and T-bet, and the molecular basis for regulating their expression, will further enhance our understanding of Th1/Th2 polarization.

We thank Andrea Wurster for critical reading of the manuscript and thoughtful discussions. J.R. is supported by a Fogarty International Fellowhip, S.J.S. is supported by a National Research Service Award, and L.H.G. is supported by NIH grants AI48126, AI31541 and AI43953 and by a Multiple Sclerosis Society grant RG2822A1.

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