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Interferon Regulatory Factor 4: Combinational Control of Lymphocyte Differentiation

Marc Veldhoen1,* 1Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK *Correspondence: [email protected] DOI 10.1016/j.immuni.2010.08.007

Transcriptional mechanisms involved in the differentiation of the recently identified interleukin-9 (IL-9) secreting T helper cell subset are still poorly defined. In this issue of Immunity, Staudt et al. (2010) now report an essential role for the interferon regulatory factor-4 in IL-9 production.

Knowledge of CD4+ T helper (Th) cell transcription (Stat) proteins, instrumental ished IL-9 production, further corrobo- biology has expanded rapidly over the in ‘‘master’’ transcription factor induction rated when IRF4-deficient T cells failed past decade. The discovery of the Th1 (Figure 1). As such, IFN-g induces Th1 to produce IL-9. Reporter gene analy- cell subset (identified via the production cells, IL-4 generates Th2 cells, and the sis and chromatin immuneprecipitation of interferon-g [IFN-g]), and the Th2 combination of IL-6 and transforming (ChIP) assays were able to determine subset (producing interleukin [Il]-4, -5, -6, growth factor (TGF)-b drives RORgt a direct interaction between IRF4 and and -13), was followed by the identifica- expression resulting in Th17 cell develop- the Il9 promoter, in contrast with an earlier tion of their respective lineage regulators. ment. The Th9 subset requires the report, which also reported diminished Th1 cell development and IFN-g pro- concerted action of both IL-4 and TGF-b IL-9 expression in retroviral-expressing duction crucially depend on the T-box (Dardalhon et al., 2008; Veldhoen et al., IRF4-transfected Th2 cells (Ahyi et al., family transcription factor Tbx21, whose 2008). 2009). ectopic expression transactivates the Gene expression involves numerous The physiological role of IL-9 is cur- IFN-g locus. Similarly, the GATA-type regulatory elements, many of which rently not well defined. Its expression is zinc finger Gata3 is necessary and suffi- require a complex of transcription factors found in allergic asthma and its produc- cient for Th2 cell differentiation and cyto- acting synergistically. The IRF family tion contributes to worm expulsion, but kine expression. The prototypic Th1 and members participate in a variety of immu- the role of IL-9 in these processes seems Th2 cell cytokines inhibit each other’s nological events, from pathogen recogni- neither required nor sufficient. However, development, whereas their master regu- tion to hematopoietic differentiation and studies in IL-9-deficient mice have estab- latory genes repress the opposing differ- immune modulation. Their involvement in lished a role in mast cell proliferation, entiation program. This suggests a strict such a wide range of processes is reflec- pulmonary goblet cell hyperplasia, and division of labor among the transcription tive of their capacity to form heterodimers mucus production. Staudt et al. use a T cell factors. This view was further strength- with each other, as well as with members adoptive transfer model to establish that ened when newly discovered subsets of other transcription factor families. The both Th2 and Th9 cell-induced airway were shown to use unique lineage deter- expression of IRF4 is restricted to the hypersensitivity reactions are very sim- mining factors: regulatory T cells (Treg) lymphoid lineage, and it is involved in ilar. However, only the Th9 cell-mediated using the FOX protein family member various processes including cytokine disease could be ameliorated via IL-9 Foxp3 and Th17 cells using the orphan signaling and expression, cell prolifera- neutralization, indicating that a Th9 cell receptor RORgt. Although a lineage tion, and apoptosis. Its deficiency results is a separate and stable subset in vivo, determining transcription factor for Th9 in lymphadenopathy, failure to produce not reverting back to Th2 cells, but also cells (producing IL-9) has not been identi- antibodies, and impaired T cell functions. confirming the redundancy of IL-9 in fied, Staudt et al. report an essential role IRF4 expression in T cells has been shown this system. IRF4-deficient cells were for the interferon regulatory factor (IRF)-4 to contribute to development of Th2, not used in this transfer model; instead, in IL-9 expression. Th17, and possibly Th1 cells, and some Irf4À/À hosts were challenged and shown The most important determinants for particular functions of Treg cells (Bru¨ stle to be resistant to disease, reflecting the lineage commitment are soluble fac- et al., 2007; Lohoff et al., 2002; Zheng impaired differentiation of both Th2 and tors such as cytokines and chemokines, et al., 2009). Comparative expression Th9 cells. released by innate immune cells and analysis of in vitro-generated Th1, Th2, The involvement of IRF4 in the devel- stromal cells upon encounter with micro- and Th9 cells revealed the presence of opment of all currently known Th cell organisms or stress. Lineage-specific sig- IRF4 in all three subsets, with highest subsets, with the possible exception of nals are largely derived from cytokine expression found in Th9 cells (Staudt Th1 cells, suggests the function of IRF4 receptor triggering. For example, binding et al., 2010). The importance of IRF4 could be upstream of lineage determina- of cytokines results in activation of partic- was established when silencing it in tion or be reliant on lineage-specific ular signal transducers and activators of established Th9 cells resulted in dimin- binding partners. The direct role of IRF4

Immunity 33, August 27, 2010 ª2010 Elsevier Inc. 141 Immunity Previews

Th1 Th2 Treg Th17 Tfh Th9

Il4, Il5, Il6, Il13 Irf4 Il4, Il5, Il6, Il13 Ifng Irf4 Icos GATA3 PU.1 IRF-4 Gata3 Il9 Irf4 Gfi1 Icos Rora, Rorc PU.1 Tbx21 Foxp3 Irf4 Bcl6 IRF-4 IRF-4 Irf4 IRF-4 Sfpi1 Stat4 Stat5 Foxp3 ? Stat3 Stat3 Irf4 Stat6 NFAT Bcl-6 Stat1 Stat6 β IL-6 TCR TGF- IL-21 IL-4, TGF-β IFN-γ IL-4 IL-21

Figure 1. Role and Interactions of IRF4 in T Helper Cell Subsets IRF4 is expressed in all known Th cell subsets. Its expression, at least in part, can be regulated via signaling strength and duration, STAT proteins, and T helper cell lineage-specific transcription factors. It is subsequently able to interact with subset-specific binding partners, like Foxp3 (Treg), Bcl6 (Tfh, Th2), and Gata3 and PU.1 (Th9), among others. The binding partner ultimately determines whether IRF4 acts in an activating or repressing manner. Staudt et al. report the importance of IRF4 in Il9 gene activation. Earlier reports indicate this may occur when partnered with PU.1. in IL-9 regulation seems to suggest ering its involvement in Icos expression, The expression of specific transcrip- the latter (Staudt et al., 2010). Interest- IL-21 responsiveness, and interaction tion factors is secondary to T cell receptor ingly, in Treg cells, Foxp3 is reported with B cell lymphoma (Bcl)-6. (TCR) activation and expansion. This to induce the expression of IRF4 (Fig- It remains untested whether ectopic is instigated upon naive T cell recogni- ure 1), whereas IRF4 deficiency does expression of IRF4 would result in the tion of cognate antigen presented by not change Foxp3 expression on a per expression of IL-9 or any other Th cell- antigen-presenting cells (APCs). Impor- cell basis. The functional defects in associated cytokines. A recent publica- tantly, it takes place in context of IRF4-deficient Treg cells are explained tion suggested that PU.1 is required additional signals, like costimulatory by the presence of composite promoter for IL-9 production as well as allergic receptors, ultimately triggering several elements for the genes encoding Foxp3 inflammation (Chang et al., 2010), in line common signal transduction cascades. and IRF4, as identified in the inducible with previous work by the same group The combination as well as the strength costimulator (Icos) gene (Figure 1). Fit- showing heterogeneous expression of and duration of these signals culminate tingly, a Foxp3-IRF4 protein complex PU.1 in Th2 cells (Chang et al., 2005). in translocation of several factors to the was indeed identified (Zheng et al., Furthermore, PU.1 was shown to directly nucleus, regulating the expression of 2009). This reflects a T cell-intrinsic func- interact with Gata3 (Chang et al., genes permissive for proliferation and tion, but its precise role is complex. IRF4 2009). Its forced expression is able to differentiation. Key determinants in this can directly activate the Il4 promoter, reduce Th2-specific cytokine expression process are nuclear factor kappa-light- and further stabilizes the Th2 cell pro- whereas its inhibition enhances Th2 cyto- chain-enhancer of activated B cells gram by influencing the expression of kine production, leaving the expression (NF-kB), activator protein 1 (AP-1), and Gfi1 (a transcriptional repressor required of IL-10 unaltered. This is in line with nuclear factor of activated T cells (NFAT) for Th2 cell differentiation) and by inter- a proposed linear relationship between proteins. A major determinant of IRF4 acting with Bcl6 (which is a known Th2 and Th9 cells in which TGF-b deviates expression is the strength of TCR stimula- repressor of the Th2 cell program). In the Th2 cell cytokine program, inhibiting tion, involving NF-kB family members. Th17 cells, IRF4 deficiency results in Il-4, Il-5, Il-6, and Il-13, toward production In addition, expression of PU.1 is also decreased expression of RORgt and of IL-9 without altering the capacity to associated with increased strength of RORa, whereas ectopic expression of produce IL-10 (Veldhoen et al., 2008). TCR stimulation, correlating with IL-10 both factors in the absence of IRF4 only Interestingly, PU.1 is a known binding production. partially rescues Th17 cell differentiation, partner of IRF4, forming a ternary com- This suggests that the earlier reported reminiscent of Stat3 deficiency (Bru¨ stle plex able to bind immunoglobulin heterogeneity within Th2 cell populations et al., 2007). IRF4 was subsequently enhancer elements. PU.1 deficiency was may reflect established Th2 cells that shown to be required for IL-21 respon- also shown to impair IL-9 production, have received varying degrees of TCR siveness, a reported stabilizer of the whereas ectopic PU.1 expression in Th2 stimulation. Increased TCR signals via Th17 cell transcriptional program sig- or Th9 cells enhanced IL-9 production NF-kB and NFAT, the latter a known naling via Stat3. In addition, IRF4 is able (Chang et al., 2010). However, the latter binding partner of IRF4, may enhance to inhibit components of the IL-1R sig- was not established in naive T cells. It the expression of both IRF4 and PU.1. naling pathway, an important component thus remains to be determined whether Additional T cell stimulation, such as for Th17 and Th9 cell activation and func- either IRF4 or PU.1 is able to enforce the with IL-2, IL-25, and IL-1b, may also tion. A role for IRF4 in yet another Th9 cell transcriptional program in naive contribute to increased expression of specialized subset of Th, follicular helper T cells (Chang et al., 2010; Staudt et al., IRF4 and PU.1. Current results thus T (Tfh) cells, seems highly likely, consid- 2010). seem indicative of Th2 cell ancestry for

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Th9 cells. The Th2 cell lineage-deter- REFERENCES Dardalhon, V., Awasthi, A., Kwon, H., Galileos, G., mining events, Stat6 signaling and Gao, W., Sobel, R.A., Mitsdoerffer, M., Strom, T.B., Elyaman, W., Ho, I.C., et al. (2008). Nat. Immunol. Ahyi, A.N., Chang, H.C., Dent, A.L., Nutt, S.L., and Gata3 expression, seem a developmental 9, 1347–1355. Kaplan, M.H. (2009). J. Immunol. 183, 1598–1606. requirement for Th9 cells. The combina- Lohoff, M., Mittru¨ cker, H.W., Prechtl, S., Bischof, tional expression of both IRF4 and PU.1 Bru¨ stle, A., Heink, S., Huber, M., Rosenpla¨ nter, C., S., Sommer, F., Kock, S., Ferrick, D.A., Duncan, could thus be instrumental in (re)program- Stadelmann, C., Yu, P., Arpaia, E., Mak, T.W., G.S., Gessner, A., and Mak, T.W. (2002). Proc. Kamradt, T., and Lohoff, M. (2007). Nat. Immunol. Natl. Acad. Sci. USA 99, 11808–11812. ming classical Th2, inhibiting IL-4, IL-5, 8, 958–966. IL-6, and IL-13 and inducing IL-9 while Staudt, V., Bothur, E., Klein, M., Lingnau, K., maintaining IL-10 (Figure 1). Chang, H.C., Zhang, S., Thieu, V.T., Slee, R.B., Reuter, S., Grebe, N., Gerlitzki, B., Hoffmann, M., Bruns, H.A., Laribee, R.N., Klemsz, M.J., and 33 The abundance of IRF4 interacting Ulges, A., Taube, C., et al. (2010). Immunity , Kaplan, M.H. (2005). Immunity 22, 693–703. this issue, 192–202. partners, determining its DNA binding interactions and its functioning as either Chang, H.C., Han, L., Jabeen, R., Carotta, S., Nutt, Veldhoen, M., Uyttenhove, C., van Snick, J., S.L., and Kaplan, M.H. (2009). J. Immunol. 183, Helmby, H., Westendorf, A., Buer, J., Martin, B., a transcriptional activator or repressor, 4887–4894. Wilhelm, C., and Stockinger, B. (2008). Nat. Immu- suggests that its interactions in different nol. 9, 1341–1346. cell types and its level of expression may Chang, H.C., Sehra, S., Goswami, R., Yao, W., Yu, Q., Stritesky, G.L., Jabeen, R., McKinley, C., Ahyi, Zheng, Y., Chaudhry, A., Kas, A., deRoos, P., Kim, be more important than the presence of A.N., Han, L., et al. (2010). Nat. Immunol. 11, J.M., Chu, T.T., Corcoran, L., Treuting, P., Klein, U., IRF4 in itself. 527–534. and Rudensky, A.Y. (2009). Nature 458, 351–356.

The Hunt for iNKT Cell Antigens: a-Galactosidase-Deficient Mice to the Rescue?

Luc Van Kaer1,* and Sebastian Joyce1 1Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA *Correspondence: [email protected] DOI 10.1016/j.immuni.2010.08.009

The endogenous lipids that control the functions of invariant natural killer T (iNKT) cells remain enigmatic. In this issue of Immunity, Darmoise et al. (2010) report that lysosomal a-galactosidase A destroys self-anti- gens recognized by iNKT cells.

Autoreactivity in the adaptive immune Following TCR engagement, iNKT cells initial study with a cell line deficient in system is normally curtailed by rigorous rapidly elicit their effector functions to b-glucosylceramide synthase pointed selection processes in central lymphoid generate protective immunity against toward glycosphingolipids as natural organs, followed by tolerance mecha- pathogens and tumors and to suppress ligands of iNKT cells (Stanic et al., 2003). nisms in the periphery. However, some autoimmunity. Several foreign lipid anti- A subsequent study demonstrated populations of lymphocytes appear to gens that can activate iNKT cells have defective iNKT cell development in mice require at least some level of autoreactiv- been identified, including the marine lacking the b-hexosaminidase A and B ity in order to effectively dispatch their sponge-derived glycosphingolipid a-gal- (b-Hex-AB) enzymes, which participate effector functions. These cells largely actosylceramide that is commonly em- in the degradation of glycosphingolipids carry out functions related to the detec- ployed to study iNKT cells. However, the in lysosomes (Zhou et al., 2004). Isoglobo- tion of self-ligands that are induced in natural self-antigens that mediate iNKT trihexosylceramide (iGb3) was identified injured or stressed tissues. Invariant cell autoreactivity remain enigmatic, and as a natural self-antigen by investigating natural killer T (iNKT) cells belong to this their identity is hotly debated (Gapin, the capacity of various products of this family of innate lymphocytes that exhibit 2010). A provocative study by Darmoise pathway to activate iNKT cells. However, natural autoreactivity. iNKT cells are et al. (2010) in this issue of Immunity subsequent studies cast doubt on iGb3 specific for lipid antigens bound by the provides evidence that the lysosomal as the sole endogenous antigen respon- MHC class I-related protein CD1d. These enzyme a-galactosidase A (a-Gal-A) sible for iNKT cell development because cells express a semi-invariant T cell controls the generation of self-antigens conventional biochemical and mass receptor (TCR) (Va14-Ja18 in mice or for iNKT cells. spectrometry techniques were unable to Va24-Ja18 in humans) together with The hunt for natural iNKT cell antigens identify iGb3 in lymphoid tissues, mice lineage markers of natural killer (NK) cells. has taken several twists and turns. An deficient in iGb3 synthase exhibited

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