ne w s and v ie w s the various aspects of the adaptive immune response. However, in moving forward, many issues will need to be addressed. Potentially the greatest hurdle is the lack of evolutionary sequence conservation, which makes it difficult to extrapolate from animal models to humans. This will need to be addressed through the development of more appropriate algorithms for the identification of the protein-binding structures and/or the short RNA-DNA pairing regions that are linked to lncRNA action9. It will also be important to develop moreefficient and high-throughput approaches for the identification of the functional lncRNAs among the thousands that are characteristically identified by high-throughput sequencing.

In this context, inhibition or overexpression of lncRNA at the transcriptional level through the use of clustered regularly interspaced palindromic repeats and a catalytically inactive form of Cas9, a nuclease associated with such repeats, might provide a novel alternative to the use of small interfering RNA or antisense oligonucleotides12 and has already been used to knock down six common lncRNAs13. In summary, the journey to understanding the role of lincRNAs and the other lncRNAs in the immune response has only just begun. However, if the functions of the various lncRNA families turn out to be as varied as those of microRNAs, there will be many exciting discoveries ahead.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. O’Connell, R.M., Rao, D.S. & Baltimore, D. Annu. Rev. Immunol. 30, 295–312 (2012). 2. Ranzani, V. et al. Nat. Immunol. 16, 318–325 (2015). 3. Djebali, S. et al. Nature 489, 101–108 (2012). 4. Guttman, M. et al. Nature 458, 223–227 (2009). 5. Heward, J.A. & Lindsay, M.A. Trends Immunol. 35, 408–419 (2014). 6. Atianand, M.K. & Fitzgerald, K.A. Trends Mol. Med. 20, 623–631 (2014). 7. Hu, G. et al. Nat. Immunol. 14, 1190–1198 (2013). 8. Gomez, J.A. et al. Cell 152, 743–754 (2013). 9. Ulitsky, I. & Bartel, D.P. Cell 154, 26–46 (2013). 10. Ho, I.C. et al. J. Exp. Med. 188, 1859–1866 (1998). 11. Geisler, S. & Coller, J. Nat. Rev. Mol. Cell Biol. 14, 699–712 (2013). 12. Bassett, A.R. et al. eLife 3, e03058 (2014). 13. Gilbert, L.A. et al. Cell 159, 647–661 (2014).

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Profiling the diversity of innate lymphoid cells Andreas Diefenbach Genome-wide transcriptional profiling of tissue-resident innate lymphoid cells (ILCs) has provided important insight not only into their developmental relationships and phenotypic plasticity but also into previously unknown functions.

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nnate lymphoid cells (ILCs) are a group of innate lymphocytes that includes conventional natural killer cells (cNK cells), lymphoid tissue inducer–like cells and various ILC subsets. ILCs are subdivided into two major branches, with cNK cells constituting the cytotoxic arm of ILCs, and helper-like ILCs represented by transcription factor T-bet–expressing ILCs of group 1 (ILC1 cells), GATA-3hi ILCs of group 2 (ILC2 cells) and RORγt+ ILCs of group 3 (ILC3 cells)1. Interestingly, the transcriptional and effector programs of the various helper-like ILC lineages largely mirror those of the previously identified helper T cell subsets, and related roles have been assigned to ILCs and helper T cells in immunity to infection and in the pathogenesis of various diseases. The finding that helper T cells and helper-like ILCs produce the same cytokines has led to an unprecedented reassessment of the unique contributions that ILCs and helper T cells make to immunity to infection and to the pathogenesis of immune system–mediated diseases. While the overall ‘layout’ of ILC lineages is now supported by robust data, a comprehensive map of their differentiation states and of their relationships is not yet Andreas Diefenbach is with the Research Centre for Immunology and the Institute of Medical Microbiology and Hygiene, University of Mainz Medical Centre, Mainz, Germany. e-mail: [email protected]

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available. In this issue of Nature Immunology, Robinette et al. report results obtained, with the support of the Immunological Genome Project Consortium, by comprehensive transcriptional profiling of almost all known ILC subsets from the small intestine (cNK cells, ILC1 cells, ILC2 cells, NKp46– ILC3 cells and NKp46+ ILC3 cells), liver (cNK cells and ILC1 cells) and spleen (cNK cells and ILC1 cells) and provide many new insights into their shared and unique transcriptional circuitry, their lineage plasticity and lineage relationships and genes previously not associated with ILC biology that will allow the exploration of functions not previously assigned to ILCs2. While high-quality transcriptional data from various published studies are already available, a comprehensive, side-byside analysis of almost all known ILC subsets has been lacking. Therefore, these data will be an invaluable repository for all ILC researchers. It is now believed that ILC identity may be controlled in part by the transcriptional regulator Id2, an inhibitor of E protein–mediated transcription, which may coordinate a core transcriptional signature of all ILCs1. The present study confirms high expression of Id2 in all ILCs, including cNK cells2. While all ILCs express Id2, two studies have suggested that all helper-like ILCs are derived from an Id2-expressing common innate lymphoid progenitor cell that cannot differentiate into cNK cells: the common helper-like

ILC progenitor3,4. These data indicate that helper-like ILCs may have a common transcriptional program distinct from that of cNK cells. Interestingly, Robinette et al. report that helper-like ILCs express a core signature of genes that is distinct from that expressed by cNK cells2. Among the genes with the highest expression in helper-like ILCs compared with their expression in cNK cells is that encoding the T cell antigen receptor (TCR) γ-chain variable region 3 (Tcrg-V3), confirmed by PCR to be a germline transcript (Fig. 1). Helper-like ILCs do not express functional γδ TCR proteins but probably have open chromatin at the Tcrg locus, which may be driven by signaling via interleukin 7 (IL-7) and/or IL-15 (ref. 5). Notably, the gene encoding the receptor for IL-7 (Il7r) is another whose expression is characteristic of all helper-like ILCs compared with its expression in cNK cells. From an evolutionary perspective, it is interesting that helper-like ILCs already make use of components of the adaptive immune system, such as the RAG recombinase. Published data have shown that components of the RAG recombinase are required for the cellular fitness of ILCs6. Future research should investigate the role of IL-7-induced chromatin remodeling and of RAG proteins in the maintenance, differentiation and memorylike qualities of ILCs. The analysis of genes uniquely expressed by individual ILC subsets suggests previously

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ne w s and v ie w s ILCs Organ environment

Helper-like ILC signature Tcrg-V3, Tmem176a/b, Il7r, Cxcr6

Differentiation niche cNK cell

Previous stimulation

ILC1

ILC2

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External stimuli (microbiota, nutrients, etc.)

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NKp46+ ILC3 cells cNK cell

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Eomes

Signature shared by all NKp46+ ILC Tbx21, Il12rb, Ifng

RORγthi RORγtlo

NK cell signature Eomes, Itgam, Klra

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ILC1 signature Tnfsf10, Tnf,Itga1, Tnfrsf10

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NK cell signature Eomes, Itgam, Klra

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ILC1 signature Tnfsf10, Tnf, Itga1, Tnfrsf10

Spleen NKp46– ILC3 cells

© 2015 Nature America, Inc. All rights reserved.

ILC2

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Liver

cNK cell

ILC1 signature Gpr55, Trat1, Mmp9, Cpne7

CD4+ ILC2 signature Il4, Il5, Il13, Il9r, Il17rb, Ccr8, Ccl1, Bmp7, Bmp2 Rxrg, Pparg, Mc5r, Dgat2, Alox5

+

NKp46 ILC3

CD4–

NKp46- ILC3 signature Ccr6, Cxcr5, Gucy1a3, Cntn1, Slc6a7, Cacna1g, Nrp1

Small intestine Signature shared by ILCs from the small intestine Rora, Atf3, Maff, Epas1, Bhlhe40, Per1, Cd69, Nr4a1

Figure 1 Transcriptional signatures of ILC subsets. ILCs are ‘preferentially’ represented at barrier surfaces, and their gene-expression program is shaped by external and organ-specific cues. Transcriptional profiling of ILCs in various organs has revealed not only specific gene-expression signatures for ILC subgroups but also transcripts shared by all ILCs in a given organ.

unknown functions for these cells. For example, the group of genes uniquely expressed by ILC2 cells includes not only familiar ones such as Il4, Il5, Il13 and Il17rb but also a cluster of genes encoding molecules involved in lipid homeostasis (i.e., Rxrg, Pparg, Mcr5, Dgat2 and Alox5) (Fig. 1). This finding is interesting in the context of two reports demonstrating important roles for ILC2 cells in the differentiation of beige fat and in limiting obesity7,8. The finding that ILC2 cells are involved in adipocyte differentiation fits nicely with the finding that ILC subsets are ‘preferentially’ represented in the small intestine2, an organ that deals not only with nutrient extraction and metabolism but also with nutrientinflicted damage. Various studies have already provided evidence that ILC function can be shaped by micronutrients9,10. An important avenue for future research will be exploring the functions of ILCs in the context of nutrient uptake and metabolic diseases. The analysis of genes uniquely expressed by NKp46– ILC3 cells identifies various genes encoding products with documented roles in the development and function of the neural system or in axonal guidance (for example, Gucy1a3, Cntn1, Slc6a7, Cacna1g

and Nrp1)2. Moreover, in ILC2 cells, two transcripts identified (Bmp7 and Bmp2) encode products known to modulate intestinal peristalsis by binding to receptors for bone-morphogenetic proteins on enteric neuronal cells. These findings add to published data demonstrating crosstalk between neuronal signaling pathways and immunological signaling pathways. Among those data was the finding that nociceptive sensory neurons regulate ‘type 17’ inflammatory responses that can be coordinated by ILC3 cells11. Future research will need to elucidate the full extent of immune system–nervous system crosstalk and to link such crosstalk to behavior, immunological function and various forms of diseases. ILCs and, in particular, helper-like ILCs are tissue-resident cells that are not well represented in secondary lymphoid organs. However, in the lamina propria of the small intestine, the various ILC subsets outnumber B cells. Robinette et al. identify organ-specific transcriptional profiles both on the ILC population level and by comparing distinct ILC lineages across various organs2. To be able to fulfill their physiological roles, ILCs may need to adapt to their organ microenvironment.

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Such phenotypic plasticity is reflected in their gene-expression program. For example, pairwise comparison of all ILC subsets of the lamina propria of the small intestine with those residing in the liver and spleen reveals a 35-transcript signature that prominently showcases the transcription factor–encoding genes Rora, Atf3, Nr4a1, Maff, Epas1, Bhlhe40 and Per1 (Fig. 1). It will be useful to identify the molecular cues that drive such organspecific plasticity. Finally, the study by Robinette et al. sheds important light on common and distinct transcriptional programs of ILC subsets that are characterized by expression of the NK cell markers NKp46 and NK1.1 (ref. 2). These data are useful for clarifying some of the controversies surrounding whether ILC1 cells and cNK cells are separate ILC lineages. In extension of a published study3, Robinette et al. show by hierarchical clustering that ILC1 cells cluster separately from cNK cells from the same organ2. Nevertheless, all NKp46+ ILCs share a signature characterized by high expression of Tbx21 (which encodes the transcription factor T-bet) and of some genes that are targets of T-bet (for example, Il12rb and Ifng). NK cells are the only subset that expresses eomesodermin, which may thus be a good marker for the separation of cNK cells from ILC1 cells. Nevertheless, this study does not resolve the issue of whether cNK cells and cells are separate ILC lineages. One limitation is that the sorting strategy used does not allow distinct separation of the various NKp46+ ILC subsets; for example, CD127neg– loNK1.1 + cell populations may include formerly RORγt+ NKp46+ ILC3 cells. Moresophisticated reporter alleles are probably needed to investigate the transcriptional program of the distinct NKp46-expressing ILC subsets. Nevertheless, it is noteworthy that three ILC types have high expression of T-bet and converge on a related transcriptional and functional program. This may indicate that T-bet-coordinated transcriptional circuitry has important functions in the immunological protection of the mucosal barrier. Although this work2 represents an important step in understanding the transcriptional networks that control the fate ‘decisions’ and functions of ILCs, there are several issues that remain to be explored. Understanding of the transcriptional programs that control ILC lineage differentiation and ILC plasticity is still very limited. This concern is of particular relevance for barrier surfaces, where complex combinations of stimuli occur that may ‘plastically’ shape the transcriptional signatures of ILCs (Fig. 1). In addition, conventional, populationbased transcription-profiling approaches may 223

ne w s and v ie w s underestimate the complexity of ILC populations. It is likely that each ILC subset contains cells of different stimulatory history, of distinct maturation states and even from different ontogenic waves of ILC development. Future work, probably requiring novel and unconventional approaches, will need to address these important challenges. At the moment, this greatly limits the ability to make predictions about the biological consequences of the observed differences in gene expression. Finally, genetic variability is not addressed in this study2. Genetic variability is frequently associated with genomic elements that

control transcription (such as enhancers), as has been demonstrated for macrophages12. It is likely that genetic variation at genomic regulatory elements, like that found in various inbred mouse strains, may make major contributions to the functional and transcriptional diversity of ILCs. Thus, the complexity of the ILC transcriptional profiles remains to be analyzed in the context of individual genetic diversity. Clearly, the brave new world of ILCs awaits additional exploration. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests.

1. Diefenbach, A., Colonna, M. & Koyasu, S. Immunity 41, 354–365 (2014). 2. Robinette, M.L. et al. Nat. Immunol. 16, 306–317 (2015). 3. Klose, C.S. et al. Cell 157, 340–356 (2014). 4. Constantinides, M.G., McDonald, B.D., Verhoef, P.A. & Bendelac, A. Nature 508, 397–401 (2014). 5. Ye, S. et al. Immunity 11, 213–223 (1999). 6. Karo, J.M., Schatz, D.G. & Sun, J.C. Cell 159, 94–107 (2014). 7. Lee, M.W. et al. Cell 160, 74–87 (2015). 8. Brestoff, J.R. et al. Nature doi:10.1038/nature14115 (22 December 2014). 9. Kiss, E.A. et al. Science 334, 1561–1565 (2011). 10. van de Pavert, S.A. et al. Nature 508, 123–127 (2014). 11. Riol-Blanco, L. et al. Nature 510, 157–161 (2014). 12. Gosselin, D. et al. Cell 159, 1327–1340 (2014).

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It takes CK2 to suppress TH2 Deepali V Sawant & Alexander L Dent Optimal immunosuppression by regulatory T cells (Treg cells) relies on gene-expression and signaling modules that are customized to the target cell. The kinase CK2 is upregulated in Treg cells and controls a newly identified Treg cell subset that acts on dendritic cells to suppress T helper type 2 inflammatory responses in the lungs.

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egulatory T cells (Treg cells) face the arduous task of maintaining steadystate immunotolerance while also suppressing exuberant inflammatory responses to commensal microorganisms, allergens and pathogens. They execute these responses via a large number of suppressive pathways. However, rather than using a ‘generic’ combination of suppression mechanisms in each context, Treg cells exhibit functional plasticity and heterogeneity, integrating cues from diverse and dynamic microenvironments to synchronize their cellular machinery with their targets. In this issue of Nature Immunology, Ulges et al. highlight a specific protein kinase, casein kinase 2 (CK2), as a member of the Treg cell arsenal that specifically controls their ability to suppress allergic T helper type 2 (T H2) responses in the lungs1. This new pathway fits with other examples in which Treg cells use context-dependent regulatory modules, such as the requirement for the transcription factors T-bet, IRF4 and STAT3 for suppression of the TH1, TH2 and TH17 subsets of helper T cells, respectively2–4.

Deepali V. Sawant is in the Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. Alexander L. Dent is in the Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA. e-mail: [email protected] or [email protected]

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Ulges and colleagues perform a comparative kinome analysis that reveals ‘preferential’ upregulation of CK2 (encoded by Csnk2) in both mouse Foxp3+ Treg cells and human Foxp3+ Treg cells following activation via the T cell antigen receptor (TCR). Genetic ablation of this kinase’s β-subunit (CK2β) in the Treg cell lineage (Csnk2bfl/flFoxp3-Cre mice) does not affect thymic T cell development or Treg cell numbers in the thymus or periphery. Despite this unaltered Treg cell homeostasis, Csnk2bfl/fl Foxp3-Cre mice develop splenomegaly and lymphadenopathy and succumb to spontaneous lung inflammation comparable to that seen in scurfy mice deficient in the transcription factor Foxp3. Activated T cells in the lung infiltrates and tracheal lymph nodes of Csnk2bfl/fl Foxp3-Cre mice exhibit hallmarks of a TH2 bias: large percentages of GATA-3+CD4+ T cells and GL7+Fas+ germinal center B cells, and high concentrations of IL-4 and serum immunoglobulin E. In fact, the baseline lung pathology of the Csnk2bfl/flFoxp3-Cre mice resembles the inflammation observed in wild-type mice following experimental induction of asthma. This selective dysregulation of the TH2 arm of the immune response in Csnk2bfl/flFoxp3-Cre mice suggests a specific role for CK2 in the control of TH2 inflammatory responses by Treg cells. Profiling by next-generation RNA sequencing in search of the molecular mechanisms underlying such dysregulation of TH2 responses reveals substantial upregulation of the inhibitory receptor ILT3 (encoded by Lilrb4) in CK2β-deficient Treg cells. Using mixed–bone

marrow chimeras and pharmacological inhibitors specific to CK2, the authors demonstrate that ILT3 is an intrinsic target of CK2-mediated regulation in Treg cells: CK2 activity inhibits ILT3 expression, such that ablation of CK2 leads to the expansion of an ILT3+ Treg cell subpopulation. The proportion of ILT3+ Treg cells increases in TH2 inflammatory environments associated with mouse models of asthma and helminth infection and in human patients with varying grades of allergies. These findings link ILT3+ Treg cells to defective control of TH2 responses. Ulges et al. verify that idea by showing that CK2β-deficient Treg cells fail to inhibit expression of the TH2 transcription factor GATA-3 in CD4+ T cells or to limit TH2 cell development but are as competent as their wild-type counterparts in controlling TH1 effector cells, both in vitro and in vivo. Strikingly, CK2β deficiency in Treg cells enhances the development of PD-L2+IRF4+ dendritic cells (DCs), a DC subset that promotes TH2 responses5,6. This ability to favor the proliferation or differentiation of TH2 response–inducing DCs might explain the selective failure of CK2β-deficient Treg cells to suppress TH2 responses. Overall, these findings suggest that in the absence of CK2β function, Treg cells upregulate ILT3 expression, which endows them with the potential to prime rather than suppress TH2 responses, via the induction of PD-L2+IRF4+ DCs (Fig. 1). How does CK2 compare with other molecular mediators linked to the control of TH2 inflammation by Treg cells, such as IRF4, Bcl-6

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Profiling the diversity of innate lymphoid cells.

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