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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">04406</article-id><article-id pub-id-type="doi">10.7554/eLife.04406</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group><subj-group subj-group-type="heading"><subject>Immunology</subject></subj-group></article-categories><title-group><article-title>The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" equal-contrib="yes" id="author-18054"><name><surname>Yu</surname><given-names>Xiaofei</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-18055"><name><surname>Wang</surname><given-names>Yuhao</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-18056"><name><surname>Deng</surname><given-names>Mi</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-18057"><name><surname>Li</surname><given-names>Yun</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-13610"><name><surname>Ruhn</surname><given-names>Kelly A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-18058"><name><surname>Zhang</surname><given-names>Cheng Cheng</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-13514"><name><surname>Hooper</surname><given-names>Lora V</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff4"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Immunology</institution>, <institution>University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Physiology</institution>, <institution>University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Developmental Biology</institution>, <institution>University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution>Howard Hughes Medical Institute, University of Texas Southwestern Medical Center</institution>, <addr-line><named-content content-type="city">Dallas</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Rath</surname><given-names>Satyajit</given-names></name><role>Reviewing editor</role><aff><institution>National Institute of Immunology</institution>, <country>India</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>Xiaofei.Yu@utsouthwestern.edu</email> (XY);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>lora.hooper@utsouthwestern.edu</email> (LVH)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>13</day><month>10</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e04406</elocation-id><history><date date-type="received"><day>19</day><month>08</month><year>2014</year></date><date date-type="accepted"><day>10</day><month>10</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Yu et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Yu et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife04406.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04406.001</object-id><p>Innate lymphoid cells (ILCs) are recently identified lymphocytes that limit infection and promote tissue repair at mucosal surfaces. However, the pathways underlying ILC development remain unclear. Here we show that the transcription factor NFIL3 directs the development of a committed bone marrow precursor that differentiates into all known ILC lineages. NFIL3 was required in the common lymphoid progenitor (CLP), and was essential for the differentiation of αLP, a bone marrow cell population that gives rise to all known ILC lineages. Clonal differentiation studies revealed that CXCR6<sup>+</sup> cells within the αLP population differentiate into all ILC lineages but not T- and B-cells. We further show that NFIL3 governs ILC development by directly regulating expression of the transcription factor TOX. These findings establish that NFIL3 directs the differentiation of a committed ILC precursor that gives rise to all ILC lineages and provide insight into the defining role of NFIL3 in ILC development.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.001">http://dx.doi.org/10.7554/eLife.04406.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.04406.002</object-id><title>eLife digest</title><p>The mucus-covered tissues that line the nose, mouth, and the digestive tract play an important role in protecting the body from infection. These mucosal tissues are the first line of defense against any pathogens we inhale or ingest, and help to keep communities of helpful bacteria—such as those that aid digestion—in place so that they can perform their beneficial functions without causing disease.</p><p>A special group of immune cells called innate lymphoid cells helps to prevent infection in the mucosal tissues and to repair damage to these tissues. There are several different types of innate lymphoid cells, with each type performing a different function. All innate lymphoid cells originate from precursor cells in the bone marrow. Some of these precursor cells had been identified previously, but were able to develop into only some of the different innate lymphoid cell types. Scientists suspected that a precursor cell existed that could develop into all types of innate lymphoid cell, but the identity of this cell had remained elusive.</p><p>Yu, Wang et al. now identify a precursor cell in the bone marrow that can produce all of the currently known different types of innate lymphoid cells. A protein called NFIL3 coaxes stem cells in the bone marrow into becoming these precursor cells, which only develop into innate lymphoid cells, and not into other immune cell types such as B cells and T cells.</p><p>Yu, Wang et al. find that NFIL3 causes some of these previously identified precursor cells to become dedicated producers of innate lymphoid cells by regulating another protein called TOX. Furthermore, gene therapy using NFIL3- or TOX-encoding DNA can help to restore normal numbers of innate lymphoid cells in mice whose bone marrow progenitor cells lack the NFIL3 gene.</p><p>These new details about how bone marrow stem cells develop into innate lymphoid cells may help scientists looking for new ways to treat infections or diseases that hamper the innate immune system.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.002">http://dx.doi.org/10.7554/eLife.04406.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>innate lymphoid cells</kwd><kwd>bone marrow progenitor</kwd><kwd>innate immunity</kwd><kwd>stem cell</kwd><kwd>intestinal immunity</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>DK070855</award-id><principal-award-recipient><name><surname>Hooper</surname><given-names>Lora V</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000861</institution-id><institution>Burroughs Wellcome Fund</institution></institution-wrap></funding-source><award-id>New Investigators in the Pathogenesis of Infectious Diseases Award</award-id><principal-award-recipient><name><surname>Hooper</surname><given-names>Lora V</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Hooper</surname><given-names>Lora V</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The transcription factor NFIL3 is essential for the development of a committed bone marrow precursor that gives rise to all known innate lymphoid cell lineages in mice.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Innate lymphoid cells (ILCs) are a recently identified family of lymphocytes that perform a variety of immune functions at barrier surfaces (<xref ref-type="bibr" rid="bib46">Spits and Cupedo, 2012</xref>). Although ILCs share a common developmental origin with B- and T-cells, they lack antigen-specific receptors. Instead, they exert their immune functions through cytokine secretion in a manner similar to T helper cells (<xref ref-type="bibr" rid="bib48">Spits et al., 2013</xref>). Despite their important contributions to immunity, the pathways that regulate ILC development remain poorly understood.</p><p>There are three known ILC groups. ILC1, which include conventional NK (cNK) cells, require the transcription factors T-BET and/or EOMES, produce interferon-γ (IFNγ) (<xref ref-type="bibr" rid="bib19">Kiessling et al., 1975</xref>; <xref ref-type="bibr" rid="bib11">Gordon et al., 2012</xref>; <xref ref-type="bibr" rid="bib8">Fuchs et al., 2013</xref>), and promote immunity to intracellular pathogens (<xref ref-type="bibr" rid="bib54">Yokoyama et al., 2004</xref>; <xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>). ILC2 require the transcription factor GATA-3, produce IL-5/13 and amphiregulin (<xref ref-type="bibr" rid="bib28">Moro et al., 2010</xref>; <xref ref-type="bibr" rid="bib30">Neill et al., 2010</xref>; <xref ref-type="bibr" rid="bib25">Monticelli et al., 2011</xref>; <xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>), and promote tissue repair and anti-helminth immunity (<xref ref-type="bibr" rid="bib26">Monticelli et al., 2012</xref>). ILC3, which include lymphoid tissue inducer (LTi) cells, depend on the transcription factor RORγt and secrete IL-17/22 (<xref ref-type="bibr" rid="bib6">Eberl and Littman, 2003</xref>; <xref ref-type="bibr" rid="bib37">Satoh-Takayama et al., 2008</xref>; <xref ref-type="bibr" rid="bib23">Luci et al., 2009</xref>; <xref ref-type="bibr" rid="bib49">Takatori et al., 2009</xref>). ILC3 are especially important for the defense of barrier surfaces as they promote anatomical containment of commensal bacteria (<xref ref-type="bibr" rid="bib44">Sonnenberg et al., 2012</xref>), regulate CD4<sup>+</sup> T cell responses to commensal bacteria (<xref ref-type="bibr" rid="bib12">Hepworth et al., 2013</xref>; <xref ref-type="bibr" rid="bib33">Qiu et al., 2013</xref>), and stimulate epithelial cells to produce antibacterial proteins (<xref ref-type="bibr" rid="bib36">Sanos et al., 2011</xref>).</p><p>All ILC differentiate from the common lymphoid progenitor (CLP), which resides in the bone marrow and also gives rise to B- and T-lymphocytes (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>; <xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>). Committed ILC progenitors that are positioned developmentally downstream of the CLP have been identified, and give rise to various ILC subsets. For example, an <italic>Id2</italic> (<underline>i</underline>nhibitor of <underline>D</underline>NA binding 2)-expressing progenitor, known as the common ‘helper-like’ innate lymphoid progenitor (CHILP), gives rise to ‘helper-like’ ILC lineages including ILC2, ILC3 and a subgroup of ILC1 (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>). PLZF-positive progenitors, termed ILCP, differentiate into non-NK ILC1, ILC2, and ILC3 (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>). However, these progenitors do not differentiate into cNK cells (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>; <xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>), suggesting that a precursor that gives rise to all ILC subtypes remains to be identified.</p><p>NFIL3 (also known as E4BP4) is a basic leucine zipper transcription factor that controls a number of different immune processes, including cytokine expression (<xref ref-type="bibr" rid="bib16">Kashiwada et al., 2011</xref>; <xref ref-type="bibr" rid="bib21">Kobayashi et al., 2011</xref>; <xref ref-type="bibr" rid="bib29">Motomura et al., 2011</xref>), IgE class switching (<xref ref-type="bibr" rid="bib17">Kashiwada et al., 2010</xref>), and T<sub>H</sub>17 cell differentiation (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). It was identified several years ago as an essential transcription factor in the differentiation of cNK cells (<xref ref-type="bibr" rid="bib9">Gascoyne et al., 2009</xref>; <xref ref-type="bibr" rid="bib15">Kamizono et al., 2009</xref>). More recently, NFIL3 has been shown also to be required for the development of non-NK ILC1 (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>), ILC2 (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>), ILC3 (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>; <xref ref-type="bibr" rid="bib22">Kobayashi et al., 2014</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>), and LTi cells (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>). Thus, NFIL3 is essential for the development of all ILC lineages.</p><p>Here we show that NFIL3 is required for the development of a common ILC progenitor from the CLP. The progenitor population is marked by CXCR6, and resides in the α<sub>4</sub>β<sub>7</sub><sup>+</sup> αLP bone marrow population, which can give rise to all ILC lineages. Clonal differentiation assays show that the CXCR6<sup>+</sup> precursors are committed ILC progenitors that differentiate into all ILC lineages but not B- or T-cells. Finally, we show that NFIL3 directs progenitor differentiation by directly regulating the expression of TOX, a known driver of ILC differentiation. These findings provide new insight into the defining role of NFIL3 in the differentiation of innate lymphoid cells.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title><italic>Nfil3<sup>−/−</sup></italic> mice are deficient in bone marrow ILC progenitors downstream of the CLP</title><p>NFIL3 has recently been shown to be essential for the development of all ILC lineages (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>). Consistent with these findings, we observed that <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice had lowered frequencies and absolute numbers of ILC2, ILC3 (including the NKp46<sup>+</sup> subtype), cNK cells, and non-NK ILC1 (<xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice also had fewer and smaller Peyer's patches in the small intestine and remaining Peyer's patches contained fewer LTi cells (RORγt<sup>+</sup> LTβ<sup>+</sup>) than wild-type mice (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>), indicating a deficiency in LTi cells that is consistent with the prior reports (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>). These data support the conclusion that NFIL3 is required for the development of all ILC lineages.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04406.003</object-id><label>Figure 1.</label><caption><title>NFIL3 is required for innate lymphoid cell development in a cell-intrinsic manner.</title><p>(<bold>A</bold>) <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice show reduced frequencies (left panel) and numbers (right panel) of major ILC types, including conventional NK (cNK), non-NK ILC1, ILC2 and ILC3. Lymphocytes were isolated from the small intestinal lamina propria and the liver and were stained as described in Materials and methods. Gating strategies are depicted in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>. cNK cells were identified as CD45<sup>+</sup> Lin(CD3ε, CD19, CD5, TCRβ, TCRγδ)<sup>-</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>+</sup>; non-NK ILC1 as CD45<sup>+</sup> Lin(CD3ε, CD19, CD5, TCRβ, TCRγδ)<sup>-</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>−</sup>; ILC2 as CD45<sup>+</sup> Lin(CD3ε, CD19)<sup>−</sup> GATA3<sup>+</sup> Sca1<sup>+</sup> KLRG1<sup>+</sup>; and ILC3 as CD45<sup>+</sup> Lin(CD3ε, CD19)<sup>-</sup> RORγt<sup>+</sup> CD127<sup>+</sup>. The NK receptor-expressing subtype of ILC3 (also known as NK22 cells) was identified by additional staining for NKp46. (<bold>B</bold>) NFIL3 regulates ILC development in a bone-marrow progenitor intrinsic manner. Equal numbers of wild-type (CD90.2<sup>+</sup> CD45.1<sup>+</sup>) and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> (CD90.2<sup>+</sup> CD45.2<sup>+</sup>) LSK cells were co-transplanted into lethally irradiated CD90.1<sup>+</sup> mice. Liver CD90<sup>+</sup> NK and non-NK ILC1 and intestinal ILC2 and ILC3 were analyzed 4-6 weeks later. The ratios of ILCs derived from wild-type (CD45.1<sup>+</sup>) and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> (CD45.2<sup>+</sup>) donor cells were calculated and plotted. Significant variation from 1.0 is indicated by *. sm. int., small intestine. (<bold>C</bold>) <italic>Nfil3</italic> regulates ILC development in a CLP-intrinsic manner. Equal numbers of wild-type (CD45.1<sup>+</sup>) and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> (CD45.2<sup>+</sup>) CLPs were co-transplanted into sublethally irradiated alymphoid <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> mice. ILCs were analyzed 4–6 weeks later as for the LSK experiment. Groups were compared by two-tailed student's t-test (<bold>A</bold>), one-sample t-test (<bold>B</bold>, LSK) or Wilcoxon signed rank test (<bold>B</bold>, CLP). Means ± SEM are shown. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.003">http://dx.doi.org/10.7554/eLife.04406.003</ext-link></p></caption><graphic xlink:href="elife04406f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Gating strategy for ILC analysis.</title><p>(<bold>A</bold>) Small intestinal lamina propria lymphocytes (LPLs) were gated on CD45<sup>+</sup> to remove non-hematopoietic cells and then on lineage markers (CD3ε and CD19) to exclude B and T cells (upper panel). ILC2 were identified as CD45<sup>+</sup> Lin<sup>−</sup> GATA3<sup>+</sup> Sca1<sup>+</sup> and were more stringently gated on expression of KLRG1. Total ILC3 were identified as CD45<sup>+</sup> Lin<sup>−</sup> RORγt<sup>+</sup> CD127<sup>+</sup> and the NKp46<sup>+</sup> ILC3 (also known as NK22) were examined by staining for NKp46. (<bold>B</bold>) Liver lymphocytes were first gated on CD45<sup>+</sup> as above and then on lineage (CD3ε, CD19, CD5, TCRβ, TCRγδ) and NK1.1. Lin<sup>−</sup> NK1.1<sup>+</sup> cells were further examined for T-BET and EOMES expression. Conventional NK (cNK) cells were identified as CD45<sup>+</sup> Lin<sup>−</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>+</sup> and non-NK ILC1 as CD45<sup>+</sup> Lin<sup>−</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>−</sup>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.004">http://dx.doi.org/10.7554/eLife.04406.004</ext-link></p></caption><graphic xlink:href="elife04406fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title><italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice are deficient in Peyer's patches and lymphoid tissue inducer cells.</title><p>(<bold>A</bold>) Peyer's patches were examined in wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice by hematoxylin and eosin (H&E) staining (upper panel) and immunofluorescence (lower panel). Anti-lymphotoxin β (LTβ) and anti-RORγt were used to detect lymphoid tissue inducer (LTi) cells and anti-B220 was used to identify B cells. Scale bars = 50 μm. (<bold>B</bold>) LTi cells were enumerated as a function of Peyer's patch area. N = 4–5 mice/group. (<bold>C</bold>) Peyer's patches were enumerated in the small intestines of wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice. (<bold>D</bold>) Peyer's patch size was measured by determining the area of the section of tissue at the center (necessitated by the irregular shape of some Peyer's patches). N = 4–5 mice/group. Statistical analysis was performed with the two-tailed student's t-test. Means ± SEM are shown. ***, p < 0.001; ****, p < 0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.005">http://dx.doi.org/10.7554/eLife.04406.005</ext-link></p></caption><graphic xlink:href="elife04406fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>LSK cells are not deficient in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice.</title><p>(<bold>A</bold>) Enrichment of Lineage-negative bone marrow cells by negative selection. Due to low frequencies of hematopoietic progenitor cells in the bone marrow of adult mice, lineage-negative cells were enriched by MACS-mediated negative selection prior to analysis or purification by FACS. To examine the efficiency of negative selection, samples before selection (upper panel) and after selection (lower panel) were subjected to CD45 and Lineage marker staining. At least 10-fold enrichment of Lineage-negative cells was routinely obtained during this process. Lineage markers used here include CD3ε, B220, CD11b, Gr-1, Ter119, CD5, TCRγδ, and NK1.1. (<bold>B</bold>–<bold>D</bold>) LSK cell frequencies and numbers are unaltered in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mouse bone marrow. Lin<sup>−</sup> cKit<sup>+</sup> Sca1<sup>+</sup> (LSK) cells roughly represent the hematopoietic stem cells (HSC). Bone marrow cells were isolated from femur and tibia from wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice. Lineage marker (Lin)-negative cells were first enriched by negative selection and then stained with antibodies against Sca1 and cKit. Typical flow plots are shown in (<bold>B</bold>) and cell frequencies and absolute cell numbers from multiple mice are pooled in (<bold>C</bold>) and (<bold>D</bold>). Comparison between genotypes was done with the two-tailed student's t-test. Means ± SEM are shown. ns, not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.006">http://dx.doi.org/10.7554/eLife.04406.006</ext-link></p></caption><graphic xlink:href="elife04406fs003"/></fig></fig-group></p><p>ILCs develop from common lymphoid progenitors (CLPs) in the bone marrow (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>; <xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>). To gain insight into the cellular origin of the broad ILC deficiency in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice, we first examined undifferentiated bone marrow precursors that were enriched by negative selection (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>). In agreement with previous findings (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>), wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> littermates harbored similar frequencies of LSK cells (Lin<sup>−</sup> Sca1<sup>+</sup> cKit<sup>+</sup>) (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>), which include hematopoietic stem cells (HSC) that give rise to all lymphoid and non-lymphoid hematopoietic cells. To test whether the requirement for NFIL3 was intrinsic to bone marrow precursors, we co-transferred wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> LSK cells into lethally irradiated mice and examined ILC subsets 5 weeks later. <italic>Nfil3</italic><sup><italic>−/−</italic></sup> LSK cells generated fewer ILC2, ILC3, and NK1.1<sup>+</sup> ILC3 in the small intestine, and fewer cNK and non-NK ILC1 in the liver (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). These data indicate that the requirement for NFIL3 in ILC development is intrinsic to bone marrow progenitors.</p><p>Like LSK cells, CLP cells are also present in normal numbers in the bone marrow of <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>). To further determine whether the NFIL3 requirement was CLP intrinsic, we co-transferred wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLP cells into sublethally irradiated alymphoid <italic>Rag2</italic><sup>−/−</sup>;<italic>Il2rg</italic><sup><italic>−/−</italic></sup> mice. 5 weeks later, <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLP had generated fewer ILC2, ILC3 in the small intestine and fewer CD90<sup>+</sup> cNK and non-NK ILC1 in the liver (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). (Because of the lower proliferation potential of CLP compared to LSK cells, progeny cells were fewer and we were not able to reliably enumerate NK1.1<sup>+</sup> ILC3 following CLP co-transfer.) These findings reveal that the requirement for NFIL3 in ILC development is intrinsic to the CLP and are consistent with the findings of Seillet et al. (<xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04406.007</object-id><label>Figure 2.</label><caption><title><italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice are deficient in bone marrow ILC precursors downstream of the CLP.</title><p>(<bold>A</bold>) <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice have comparable CLP frequencies but show deficiencies in αLP. Bone marrow cells were isolated from femur and tibia of wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice. Lineage marker (CD3ε, B220, CD11b, Gr-1, Ter-119, CD5, TCRγδ, NK1.1)-negative (Lin<sup>−</sup>) cells were first enriched by negative selection and then stained with antibodies to identify CLP (Lin<sup>−</sup> cKit<sup>low</sup> CD127<sup>+</sup> Sca1<sup>low</sup> Flt3<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>-</sup>) and αLP (Lin<sup>−</sup> cKit<sup>low</sup> CD127<sup>+</sup> Sca1<sup>low</sup> Flt3<sup>-</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup>) (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>). Gating strategy and representative flow plots are shown on the left and combined data for the frequencies and absolute numbers of CLP and αLP are shown on the right. (<bold>B</bold>) <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice are deficient in common ‘helper-like’ innate lymphoid progenitor (CHILP) cells and ILC2P (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>). Bone marrow cells were processed as above and CHILPs and ILC2Ps were identified as Lin<sup>−</sup> CD127<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD25<sup>-</sup> Flt3<sup>-</sup> and Lin<sup>−</sup> CD127<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD25<sup>+</sup> Sca1<sup>+</sup>, respectively. (<bold>C</bold>) Expression of key transcription factors involved in ILC development in CLP, αLP and CHILP. Bone marrow cells were isolated from <italic>Id2</italic><sup>GFP/+</sup> and <italic>Rorgt</italic><sup>GFP/+</sup> mice to examine Id2 and RORγt expression. Expression of GATA3, PLZF, T-BET and EOMES were examined in C57BL/6 mice with specific antibodies. Statistical analysis was performed with two-tailed student's t-test. Means ± SEM are shown. ns, not significant; ***, p < 0.001, ****, p < 0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.007">http://dx.doi.org/10.7554/eLife.04406.007</ext-link></p></caption><graphic xlink:href="elife04406f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.008</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Gating strategies for bone marrow lymphoid progenitor analysis.</title><p>(<bold>A</bold>) Gating of common lymphoid progenitor (CLP) cells and α<sub>4</sub>β<sub>7</sub> integrin-expressing CLP (αLP) in the bone marrow. Bone marrow cells were released from femur and tibia and differentiated cells were removed by negative selection. Cells were then stained with anti-biotin (Lineage), cKit, Sca1, CD127, Flt3 and α<sub>4</sub>β<sub>7</sub>. CLPs are identified as Lin<sup>−</sup> cKit<sup>lo</sup> Sca1<sup>lo</sup> CD127<sup>+</sup> Flt3<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>−</sup> and αLP as Lin<sup>−</sup> cKit<sup>lo</sup> Sca1<sup>lo</sup> CD127<sup>+</sup> Flt3<sup>−</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup>. (<bold>B</bold>) Gating of CHILP and ILC2P cells. Lineage-negative cells were prepared as above and stained with anti-biotin (Lineage), CD25, Sca1, CD127, Flt3 and α<sub>4</sub>β<sub>7</sub>. CHILPs are identified as Lin<sup>−</sup> CD127<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD25<sup>−</sup> Flt3<sup>−</sup> and ILC2P as Lin<sup>−</sup> CD127<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD25<sup>+</sup> Sca1<sup>+</sup>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.008">http://dx.doi.org/10.7554/eLife.04406.008</ext-link></p></caption><graphic xlink:href="elife04406fs004"/></fig></fig-group></p><p>The CLP gives rise to all lymphoid cells, including ILCs, T cells, and B cells. In contrast to ILC numbers, overall T and B cell numbers are not altered in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice (<xref ref-type="bibr" rid="bib17">Kashiwada et al., 2010</xref>). The general requirement for NFIL3 in ILC development therefore suggested that NFIL3 might be essential for the development of ILC-committed precursors downstream of the CLP. To further investigate the cellular origin of the ILC developmental deficiency in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice, we analyzed various bone marrow precursor populations downstream of the CLP in wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice. <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice had markedly fewer Flt3<sup>-</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CLPs (known as αLPs) (<xref ref-type="fig" rid="fig2">Figure 2A</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A</xref>), which have been shown to differentiate into ILC3 and NK cells (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>).</p><p><italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice also had fewer previously identified precursor cells that have a more restricted differentiation potential. These cells include ILC2 progenitor cells (ILC2P, Lin<sup>−</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD127<sup>+</sup> Sca1<sup>+</sup> CD25<sup>+</sup>) that only differentiate into ILC2 (<xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>) (<xref ref-type="fig" rid="fig2">Figure 2B</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>), and the CHILP that can give rise to non-NK ILC1, ILC2 and NK1.1<sup>+</sup> NKp46<sup>+</sup> ILC3 (<xref ref-type="fig" rid="fig2">Figure 2B</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>). Similarly, NFIL3 has been found to be critical for generation of the earliest NK-committed precursors (PreNKP) (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>). Thus, <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice have reduced numbers of precursors that give rise to cNK cells, non-NK ILC1, ILC2 and ILC3. Together, these data indicate that NFIL3 is required for generation of ILC precursors in the bone marrow.</p></sec><sec id="s2-2"><title>αLP differentiate into all ILC lineages</title><p>It is thought that ILCs differentiate from a common ILC progenitor population (<xref ref-type="bibr" rid="bib48">Spits et al., 2013</xref>; <xref ref-type="bibr" rid="bib50">Tanriver and Diefenbach, 2014</xref>). Prior studies have identified progenitor populations that develop into most, but not all, subtypes of known ILC lineages (<xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>; <xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>; <xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>). The CHILP, identified through <italic>Id2</italic> lineage tracing studies, can differentiate into non-NK ILC1, ILC2 and NK1.1<sup>+</sup> NKp46<sup>+</sup> ILC3 but not cNK cells (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>), and the PLZF-dependent ILCP gives rise to all ILCs except cNK cells (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>). These findings accord with the partial ILC deficiencies seen in mice lacking <italic>Id2</italic> and <italic>Zbtb16</italic> (encoding PLZF) (<xref ref-type="bibr" rid="bib2">Boos et al., 2007</xref>; <xref ref-type="bibr" rid="bib38">Savage et al., 2008</xref>). In particular, cNK cell development is not impaired in <italic>Zbtb16</italic><sup><italic>−/−</italic></sup> mice, while <italic>Id2</italic><sup><italic>−/−</italic></sup> mice show cNK developmental defects only during NK maturation (<xref ref-type="bibr" rid="bib2">Boos et al., 2007</xref>). Similarly, ILC2Ps are lineage-specified progenitors of ILC2s with no appreciable potential to differentiate into cNK cells or ILC3 (<xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>). The broad ILC deficiency (including cNK cells) and impaired ILC precursor development in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice thus suggested that the NFIL3 might be required for the generation of a common ILC progenitor that lies developmentally upstream of the previously identified ILC precursors. We therefore sought to identify NFIL3-dependent precursor populations that differentiate into all ILC lineages.</p><p>In contrast to CHILP, ILCP, and ILC2P, αLP cells can differentiate into both cNK cells and ILC3 (<xref ref-type="bibr" rid="bib55">Yoshida et al., 2001</xref>; <xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>) and thus likely represent an earlier stage of ILC development. This idea is supported by expression profiles of key transcription factors known to be involved in ILC development (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). Similar to CLP and CHILP, αLPs do not express transcription factors that specify ILC lineages, such as RORγt, GATA3, T-BET and EOMES, suggesting an undifferentiated phenotype. However, in contrast to CHILPs, which uniformly express high levels of ID2, only a small fraction of αLPs are ID2<sup>+</sup> (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). The majority of αLPs express ID2 at levels that are markedly lower than those in CHILPs. Because ID2 is virtually undetectable in CLPs, this suggests that αLPs may represent a transitional stage between CLP and CHILP. This is further supported by the fact that αLPs do not express PLZF while a major fraction of CHILPs express PLZF (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>), which defines another group of ILC precursors (ILCP) that lack cNK cell differentiation potential (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>).</p><p>To determine whether αLP can also give rise to ILC2, we co-cultured purified αLP with bone marrow stromal OP9 cells (OP9-GFP) or OP9 cells expressing the Notch ligand Delta-like 1 (OP9-DL1), which support ILC differentiation in vitro (<xref ref-type="bibr" rid="bib13">Holmes and Zúñiga-Pflücker, 2009</xref>; <xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>; <xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>). When co-cultured with OP9-DL1 cells in the presence of ILC2-inducing cytokines, αLPs readily developed into ILC2 as the majority of progeny cells expressed ILC2 markers (GATA3<sup>+</sup> Sca1<sup>+</sup>) (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). When OP9-GFP cells (not expressing Notch ligand) were used in this assay, only a small fraction of progeny cells became ILC2 (<xref ref-type="fig" rid="fig3">Figure 3A</xref>), confirming that Notch signaling is important for ILC2 differentiation in vitro (<xref ref-type="bibr" rid="bib51">Wong et al., 2012</xref>; <xref ref-type="bibr" rid="bib52">Yang et al., 2013</xref>). In agreement with a prior study (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>), αLP differentiated into ILC3 and RORγt<sup>−</sup> NK1.1<sup>+</sup> cells under ILC3-inducing conditions (<xref ref-type="fig" rid="fig3">Figure 3A</xref>).<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04406.009</object-id><label>Figure 3.</label><caption><title>αLPs can differentiate into ILC2 in vitro and in vivo, and can thus give rise to all known ILC lineages.</title><p>(<bold>A</bold>) αLPs can differentiate into ILC2 in vitro. αLPs were purified by FACS and ∼25 cells were co-cultured with a bone marrow stromal cell line OP9 (OP9-GFP) or OP9 cells stably expressing the Notch ligand Delta-like 1 (OP9-DL1) for 14 days in the presence of ILC2-inducing (IL-2) or ILC3-inducing (IL-23) cytokines. Cells were then stained and analyzed by flow cytometry. ILC2 cells were identified as CD45<sup>+</sup> CD3ε<sup>−</sup> CD19<sup>-</sup> GATA3<sup>+</sup> Sca1<sup>+</sup>, ILC3 as RORγt<sup>+</sup> NK1.1<sup>-</sup>, and ILC1 (including NK and non-NK ILC1) as CD45<sup>+</sup> CD3ε<sup>−</sup> CD19<sup>-</sup> RORγt<sup>−</sup> NK1.1<sup>+</sup>. Typical flow plots are shown on the left and combined data are shown on the right. (<bold>B</bold> and <bold>C</bold>) αLPs can differentiate into ILC2, ILC3, cNK, and non-NK ILC1 in vivo. αLP cells were purified from wild-type (CD45.1<sup>+</sup>) mice and ∼1000 αLP cells were transplanted into sublethally irradiated <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> (CD45.2<sup>+</sup>) mice. ILCs in the small intestine and colon (<bold>B</bold>) or liver (<bold>C</bold>) were examined 4–6 weeks later. (<bold>D</bold>) αLPs failed to differentiate into B cells both in the small intestine and spleen. There were small numbers of T cells in both the small intestine and spleen. Statistical analysis was performed with two-tailed student's t-test. Means ± SEM are shown. N.D., not detected; *, p < 0.05; ***, p < 0.001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.009">http://dx.doi.org/10.7554/eLife.04406.009</ext-link></p></caption><graphic xlink:href="elife04406f003"/></fig></p><p>To assess the potential of αLPs to differentiate into ILC2 in vivo, we transferred ∼1000 purified αLPs (CD45.1<sup>+</sup>) into sublethally irradiated <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> mice (CD45.2<sup>+</sup>). After 5 weeks, ILC2 that had differentiated from engrafted αLPs were detected in small intestine and colon of the recipient mice (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). We noted that GATA3<sup>+</sup> ILC2 comprised a small fraction of CD127<sup>+</sup> ILCs in the small intestine but were the majority in the colon while RORγt<sup>+</sup> ILC3 showed the reverse tissue distribution pattern (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). This suggests that tissue-specific microenvironment influences ILC development or recruitment. Consistent with the previously reported cNK cell differentiation potential of αLPs (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>), donor cells gave rise to cNK cells in the liver, and also differentiated into non-NK ILC1 (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). Differentiation of ILCs from αLPs was not caused by contamination of αLPs with CLPs, as no donor-derived B cells were detected in the spleen and small intestine of recipient mice (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). This accords with the loss of B cell differentiation potential in αLPs (<xref ref-type="bibr" rid="bib55">Yoshida et al., 2001</xref>; <xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>). However, there were small numbers of donor-derived T cells, consistent with prior findings that αLP retain some T cell differentiation potential (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>). Thus, αLPs can give rise to all known ILC lineages in vitro and in vivo. Given the more restricted differentiation potential of CHILP, ILCP, and ILC2P, αLPs are therefore likely to be developmentally upstream of these progenitors, and defective αLP development in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice can thus explain the general ILC deficiency in these mice.</p></sec><sec id="s2-3"><title>CXCR6<sup>+</sup> αLP cells differentiate into all ILC lineages but not T- and B-cells</title><p>The residual T cell differentiation potential of αLPs suggested that this population includes cells that are not fully committed ILC precursors. Prior studies have shown that when αLP cells acquire CXCR6 expression, they continue to give rise to cNK cells and ILC3 but lose T cell differentiation potential (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>). CXCR6<sup>+</sup> cells comprised 3–4% of the αLP population in the bone marrows of adult wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). Their absolute numbers were diminished in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), in parallel with the decrease in total αLP numbers (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). We therefore hypothesized that CXCR6<sup>+</sup> αLPs might include NFIL3-dependent committed ILC precursors that give rise to all ILC lineages. To assess the developmental potential of CXCR6<sup>+</sup> αLP cells, we isolated cells by flow cytometry and cultured individual cells with OP9-DL1 feeder cells in the presence of non-polarizing SCF and IL-7 (<xref ref-type="fig" rid="fig4">Figure 4C,D</xref>). In contrast to CXCR6<sup>-</sup> αLP cells, which retained the ability to differentiate into T cells, CXCR6<sup>+</sup> αLP cells failed to give rise to T cells in any of the clones examined (<xref ref-type="fig" rid="fig4">Figure 4C</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.04406.010</object-id><label>Figure 4.</label><caption><title>CXCR6<sup>+</sup> αLP cells are ILC-committed precursors.</title><p>(<bold>A</bold>) αLP can be divided into two subpopulations based on CXCR6 expression. (<bold>B</bold>) Enumeration of CXCR6<sup>+</sup> cells in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice. Bone marrow progenitor cells were enriched by negative selection with Lin<sup>−</sup> cell counts ranging from 8–12.5 × 10<sup>6</sup> per mouse. Lin<sup>−</sup> cells were then stained and analyzed by flow cytometry. The numbers of CXCR6<sup>+</sup> αLP cells in 3.3 × 10<sup>6</sup> Lin<sup>−</sup> cells are plotted. (<bold>C</bold>) CXCR6<sup>+</sup> αLP cells lack T cell differentiation potential. CXCR6<sup>-</sup> and CXCR6<sup>+</sup> αLP cells were individually sorted into the wells of a 96-well plate with an irradiated OP9-DL1 feeder cell monolayer. Cells were cultured in the presence of 20 ng/ml SCF and 20 ng/ml IL-7 for 3 weeks. T cells were detected by CD3ε staining. Data are shown as the percentages of CD45<sup>+</sup> cell-containing wells in which T cells were detected. (<bold>D</bold>) CXCR6<sup>+</sup> αLP cells are multipotent precursors to cNK cells, non-NK ILC1, ILC2 and ILC3 in vitro. Individual CXCR6<sup>+</sup> αLP cells were sorted and cultured as above (240 cells in total, pooled from two independent experiments). ILCs were analyzed by flow cytometry. cNK cells were detected as CD45<sup>+</sup> CD3ε<sup>−</sup> CD19<sup>-</sup> RORγt<sup>−</sup> GATA3<sup>-</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>+</sup>; non-NK ILC1 as CD45<sup>+</sup> CD3ε<sup>−</sup> CD19<sup>-</sup> RORγt<sup>−</sup> GATA3<sup>-</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>−</sup>, ILC2 as CD45<sup>+</sup> CD3ε<sup>−</sup> CD19<sup>-</sup> GATA3<sup>+</sup>; and ILC3 as CD45<sup>+</sup> CD3ε<sup>−</sup> CD19<sup>-</sup> RORγt<sup>+</sup>. Each well is presented as a column, with detected ILC lineages highlighted in blue. (<bold>E</bold>) CXCR6<sup>+</sup> αLP cells differentiate into cNK cells, non-NK ILC1, ILC2 and ILC3 in vivo. ∼1000 CXCR6<sup>+</sup> αLP cells were purified from 20 CD45.1<sup>+</sup> mice by FACS sorting and were transplanted into sublethally irradiated <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> (CD45.2<sup>+</sup>) mice. T cells and B cells in the blood and ILCs in the small intestine and liver were examined 4–6 weeks later. Data shown are representative of two independent experiments. Statistical analysis was performed with two-tailed student's t-test. Means ± SEM are shown. **, p < 0.01.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.010">http://dx.doi.org/10.7554/eLife.04406.010</ext-link></p></caption><graphic xlink:href="elife04406f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.011</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Frequencies of CXCR6<sup>+</sup> cells in wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> αLP cell populations.</title><p>Bone marrow progenitors from wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice were enriched by negative selection and stained to detect CXCR6<sup>+</sup> αLP cells. The frequencies of CXCR6<sup>+</sup> cells among αLP cells are plotted. Statistical analysis was performed by two-tailed student's t-test. Means ± SEM are shown. ns, not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.011">http://dx.doi.org/10.7554/eLife.04406.011</ext-link></p></caption><graphic xlink:href="elife04406fs005"/></fig></fig-group></p><p>To determine if a single CXCR6<sup>+</sup> αLP cell could give rise to all ILC lineages, we assessed the clonal differentiation potential of the CXCR6<sup>+</sup> αLP cells in vitro. While no T cells were detected, mixtures of cNK, non-NK ILC1, ILC2, and ILC3 cells were present in the progeny populations of single CXCR6<sup>+</sup> αLP cells (<xref ref-type="fig" rid="fig4">Figure 4C,D</xref>). Approximately 60% of wells with clonal growth contained multiple ILC lineages. In particular, 43.3% of the wells differentiated from individual CXCR6<sup>+</sup> αLP cells contained two ILC lineages, and 11.7% three ILC lineages (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). Importantly, 2.5% of wells contained all four ILC lineages, demonstrating that individual CXCR6<sup>+</sup> αLP cells can differentiate into all known ILC lineages. In agreement with the in vitro differentiation data, CXCR6<sup>+</sup> αLP cells differentiated into cNK, non-NK ILC1, ILC2 and ILC3, but not B cells or T cells, when transferred into sublethally-irradiated <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> mice (<xref ref-type="fig" rid="fig4">Figure 4E</xref>). Thus, CXCR6<sup>+</sup> αLP cells include committed ILC precursors that can differentiate into all major ILC lineages in vitro and in vivo.</p></sec><sec id="s2-4"><title>NFIL3-dependent ILC development is mediated by Tox</title><p>To identify potential mechanisms underlying NFIL3-dependent ILC development, we isolated CLPs from wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice and surveyed their transcriptomes by Illumina BeadArrays. <italic>Nfil3</italic> expression was readily detected in CLPs (<xref ref-type="fig" rid="fig5">Figure 5A</xref>), which accords with previous reports (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>) and is consistent with the finding that NFIL3 regulates ILC development in a CLP-intrinsic manner (<xref ref-type="fig" rid="fig1">Figure 1C</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>). However, there was no detectable expression in CLPs of other transcription factors that are known to govern ILC development (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). These factors include <italic>Id2</italic> (<xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>; <xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>), <italic>Zbtb16</italic> (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>), <italic>Eomes</italic> (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>), <italic>Tcf7</italic> (encoding TCF-1) (<xref ref-type="bibr" rid="bib52">Yang et al., 2013</xref>), <italic>Rora</italic> (<xref ref-type="bibr" rid="bib51">Wong et al., 2012</xref>), <italic>Rorc</italic> (<xref ref-type="bibr" rid="bib6">Eberl and Littman, 2003</xref>; <xref ref-type="bibr" rid="bib39">Sawa et al., 2010</xref>), <italic>Gata3</italic> (<xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>) and <italic>Tbx21</italic> (<xref ref-type="bibr" rid="bib11">Gordon et al., 2012</xref>; <xref ref-type="bibr" rid="bib34">Rankin et al., 2013</xref>). In contrast, the <underline>h</underline>igh <underline>m</underline>obility <underline>g</underline>roup (HMG) transcriptional regulator <italic>Tox</italic>, which is known to regulate NK and ILC3 development (<xref ref-type="bibr" rid="bib1">Aliahmad et al., 2010</xref>), was expressed at a detectable level in wild-type CLPs and was down-regulated in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLPs (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>). This suggested that NFIL3 might regulate <italic>Tox</italic> expression in CLPs.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.04406.012</object-id><label>Figure 5.</label><caption><title>NFIL3-dependent ILC development is mediated by <italic>Tox</italic>.</title><p>(<bold>A</bold> and <bold>B</bold>) <italic>Tox</italic> expression is lower in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLPs than in wild-type cells. (<bold>A</bold>) Heatmap comparing expression levels of transcription factors in wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLPs (left). Factors included <italic>Nfil3</italic>, <italic>Tox</italic> and other transcription factors that are known to be involved in ILC development. The absolute signal values and detection p values for each transcription factor in Illumina BeadArrays are also shown (right). Note that only <italic>Nfil3</italic> and <italic>Tox</italic> expression can be reliably detected in wild-type CLP cells. (<bold>B</bold>) Q-PCR analysis of <italic>Tox</italic> expression in wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLPs. (<bold>C</bold>–<bold>E</bold>) NFIL3 activates <italic>Tox</italic> expression by binding directly to the <italic>Tox</italic> promoter. (<bold>C</bold>) <italic>Tox</italic> expression was determined by Q-PCR following shRNA knockdown of NFIL3 (left), and NFIL3 overexpression (right) in EL4 cells. (<bold>D</bold>) ChIP analysis of EL4 cells using an NFIL3-specific antibody or IgG control. <italic>Tox</italic> promoter (nt −2105 to −1867) enrichment was calculated as the ratio of the NFIL3-specific antibody pull-down to the IgG control pull-down. The left panel shows results with endogenous NFIL3 levels and the right panel shows results with NFIL3 overexpression. (<bold>E</bold>) Luciferase reporter assay. A 2.8 kb fragment of the <italic>Tox</italic> promoter was cloned and fused with the firefly <italic>luciferase</italic> gene to generate a <italic>Tox-luciferase</italic> reporter. HEK293T cells were co-transfected with the reporter and an empty vector or an NFIL3-encoding vector. Luciferase activity was normalized to cells transfected with vector-only controls. (<bold>F</bold>) Restoring <italic>Tox</italic> expression in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> progenitors rescues ILC development in vivo. <italic>Nfil3</italic><sup><italic>−/−</italic></sup> LSK cells (CD45.2<sup>+</sup>) were retrovirally transduced with either an empty vector (MSCV-IRES-hCD2), a TOX-encoding vector (MSCV-<italic>Tox</italic>-IRES-hCD2), or an NFIL3-encoding vector (MSCV-<italic>Nfil3</italic>-IRES-hCD2) and then transferred into lethally irradiated wild-type (CD45.1<sup>+</sup>) mice. ILCs were examined 5–6 weeks later. The frequencies of total ILC2 and ILC3 within CD45.2<sup>+</sup> hCD2<sup>+</sup> cells are shown. Statistical comparisons between groups were performed with two-tailed student's t-test (<bold>B</bold>–<bold>E</bold>), nonparametric one-way ANOVA test with posttests (<bold>F</bold>). Means ± SEM are shown. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.012">http://dx.doi.org/10.7554/eLife.04406.012</ext-link></p></caption><graphic xlink:href="elife04406f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.013</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Knockdown of <italic>Nfil3</italic> by shRNA.</title><p>(<bold>A</bold>) shRNA constructs were screened for <italic>Nfil3</italic> knockdown. HEK293T cells were co-transfected with an <italic>Nfil3</italic>-coding plasmid (CDS only) and shRNA constructs. NFIL3 protein levels were examined by Western blotting 36 hr later. A scrambled shRNA was used as a negative control and ACTIN was used as a loading control during Western blotting. (<bold>B</bold>) <italic>Nfil3</italic> was knocked down by shRNAs, sh39 and sh40, in EL4 cells. HEK293T cells were co-transfected with shRNA constructs, pVSVG and pCMVDR9. Cell culture supernatants were harvested 48 hr later and lentiviral particles were concentrated by ultracentrifugation. EL4 cells were transduced with shRNA-encoding lentivirus by spinoculation in the presence of 4 μg/ml polybrene. Cells stably expressing shRNAs were selected with 8 μg/ml puromycin and live cells (propidium iodide-negative) were purified by FACS. <italic>Nfil3</italic> mRNA levels were examined by Q-PCR with <italic>Gapdh</italic> as an internal control. Statistical analysis was performed with one-way ANOVA with post-tests. Means ± SEM are shown. ****, p < 0.0001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.013">http://dx.doi.org/10.7554/eLife.04406.013</ext-link></p></caption><graphic xlink:href="elife04406fs006"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04406.014</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Experimental design and gating strategy for the <italic>Tox</italic> rescue experiment.</title><p>(<bold>A</bold>) Schematic illustrating the experimental design. <italic>Nfil3</italic><sup><italic>−/−</italic></sup> LSK (CD45.2<sup>+</sup>) cells were retrovirally transduced with empty (MSCV-IRES-hCD2), TOX-encoding (MSCV-<italic>Tox</italic>-IRES-hCD2) or NFIL3-encoding (MSCV-<italic>Nfil3</italic>-IRES-hCD2) vectors. Cells were then transplanted into lethally irradiated wild-type mice (CD45.1<sup>+</sup>) and ILCs were examined 4–6 weeks later. (<bold>B</bold>) Gating strategy for examining ILCs in the recipient mice. Live cells were first electronically gated as ZombieGreen-negative and cells transduced by retrovirus were identified as CD45.2<sup>+</sup> hCD2<sup>+</sup>. ILC2, ILC3, cNK and non-NK ILC1 were gated as Lineage (CD3, CD19, CD5, TCRβ, TCRγδ)<sup>−</sup> CD127<sup>+</sup> GATA3<sup>+</sup>, Lin<sup>−</sup> CD127<sup>+</sup> RORγt<sup>+</sup>, Lin<sup>−</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>+</sup> and Lin<sup>−</sup> NK1.1<sup>+</sup> T-BET<sup>+</sup> EOMES<sup>−</sup>, respectively. The results of the experiment are summarized in <xref ref-type="fig" rid="fig5">Figure 5F</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.014">http://dx.doi.org/10.7554/eLife.04406.014</ext-link></p></caption><graphic xlink:href="elife04406fs007"/></fig></fig-group></p><p>CLPs are present in small numbers in adult mice (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>), making it challenging to perform biochemical studies of <italic>Tox</italic> regulation by NFIL3 using these cells. As an alternative, we found that NFIL3 regulates <italic>Tox</italic> expression in EL4 cells, a mouse lymphoma cell line (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). Knockdown of NFIL3 in EL4 cells with two independent shRNA constructs led to dose-dependent down-regulation of <italic>Tox</italic> expression (<xref ref-type="fig" rid="fig5">Figure 5C</xref>; <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>). Conversely, overexpression of NFIL3 in EL4 cells increased <italic>Tox</italic> expression (<xref ref-type="fig" rid="fig5">Figure 5C</xref>), indicating that <italic>Tox</italic> expression is sensitive to NFIL3 levels in EL4 cells in a manner similar to CLPs. A chromatin immunoprecipitation (ChIP) assay with an NFIL3-specific antibody (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>) demonstrated that NFIL3 directly bound to the <italic>Tox</italic> promoter (nt −2105 to −1867) and that overexpression of NFIL3 enhanced this binding (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Finally, NFIL3 activated <italic>Tox</italic> promoter activity as assessed by a luciferase reporter assay (<xref ref-type="fig" rid="fig5">Figure 5E</xref>)<italic>.</italic> Thus, NFIL3 activates <italic>Tox</italic> expression by directly binding to its promoter. EL4 cells are derived from T lymphocytes, a CLP-derived lineage, and thus we cannot exclude the possibility that the regulatory relationship between <italic>Nfil3</italic> and <italic>Tox</italic> differs between T lymphocytes and CLPs. Nevertheless, our studies on CLPs and EL4 cells both support the idea that NFIL3 is an activator of <italic>Tox</italic> expression.</p><p>Because <italic>Tox</italic> is known to be essential for cNK and ILC3 development (<xref ref-type="bibr" rid="bib1">Aliahmad et al., 2010</xref>), we postulated that lowered <italic>Tox</italic> expression leads to the broad ILC deficiency in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice and that restoring <italic>Tox</italic> expression would rescue ILC development. To test this idea, we cloned <italic>Tox</italic> coding sequences into a bicistronic vector (MSCV-IRES-hCD2), which allowed expression of the native form of TOX and also marked cells with the cell surface marker hCD2. We then delivered the TOX-encoding plasmid or the empty vector into purified <italic>Nfil3</italic><sup><italic>−/−</italic></sup> LSK cells (CD45.2<sup>+</sup>) by retroviral transduction (<xref ref-type="bibr" rid="bib57">Zheng et al., 2012</xref>; <xref ref-type="bibr" rid="bib45">Spencer et al., 2014</xref>), followed by transfer of these cells into lethally irradiated wild-type mice (CD45.1<sup>+</sup>) (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). Compared to the empty vector control, transduction of the TOX-encoding plasmid led to increased numbers of cNK cells in spleen, non-NK ILC1 cells in liver, and ILC2 and ILC3 in the small intestines of recipient mice (<xref ref-type="fig" rid="fig5">Figure 5F</xref>). We observed that rescue of ILC development from <italic>Nfil3</italic><sup><italic>−/−</italic></sup> LSK cells by <italic>Tox</italic> was largely comparable to rescue by <italic>Nfil3</italic> in the same setting, supporting the idea that <italic>Tox</italic> acts downstream of <italic>Nfil3</italic> in ILC development. Though ILC2 cells developing from <italic>Tox</italic>-rescued LSK cells were generally fewer than those from <italic>Nfil3</italic>-rescued LSK cells, the difference between the two groups was not statistically significant. Thus, ILC development is rescued by restoring <italic>Tox</italic> expression in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> progenitors, indicating that NFIL3 drives ILC development in part by regulating <italic>Tox</italic> expression.</p></sec><sec id="s2-5"><title>NFIL3-dependent ILC development is essential for host defense against Citrobacter rodentium infection</title><p>IL-22 is produced both by ILC3 and T<sub>H</sub>17 cells and is essential for protection against <italic>Citrobacter rodentium</italic> infection (<xref ref-type="bibr" rid="bib37">Satoh-Takayama et al., 2008</xref>; <xref ref-type="bibr" rid="bib58">Zheng et al., 2008</xref>). <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice show elevated susceptibility to intestinal pathogens such as <italic>Citrobacter rodentium</italic> (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>). However, <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice retain T<sub>H</sub>17 cells, which are elevated relative to wild-type mice (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). To rule out confounding effects of T<sub>H</sub>17 cells, we first crossed <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice with <italic>Rag1</italic><sup><italic>−/−</italic></sup> mice to create <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> mice, which lack T and B cells in addition to ILCs. <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> mice were more susceptible to oral <italic>C. rodentium</italic> infection than <italic>Rag1</italic><sup><italic>−/−</italic></sup> mice as measured by weight loss (<xref ref-type="fig" rid="fig6">Figure 6</xref>). These data thus suggest that NFIL3-dependent ILC development is essential for host immune defense against a mucosal pathogen.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.04406.015</object-id><label>Figure 6.</label><caption><title><italic>Nfil3</italic> deficiency results in increased susceptibility to <italic>C. rodentium</italic> infection in mice.</title><p><italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice were crossed with <italic>Rag1</italic><sup><italic>−/−</italic></sup> mice to generate <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> mice to eliminate the effects of adaptive immune cells, especially T<sub>H</sub>17 cells. <italic>Rag1</italic><sup><italic>−/−</italic></sup> and <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> mice were orally challenged with 5 × 10<sup>9</sup> CFU of <italic>C. rodentium</italic> and mouse weight loss was monitored. 4 <italic>Rag1</italic><sup><italic>−/−</italic></sup> mice and 5 <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> mice were analyzed. Comparisons were carried out with or two-way ANOVA with posttests. Means ± SEM are shown. *, p < 0.05.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04406.015">http://dx.doi.org/10.7554/eLife.04406.015</ext-link></p></caption><graphic xlink:href="elife04406f006"/></fig></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Innate lymphoid cells are essential players in the immune response to various infections and in maintenance of barrier function in mucosal tissues. ILCs arise in the bone marrow from the CLP, and thus share a common developmental origin with T- and B-cells. The pathways that govern ILC differentiation downstream of the CLP have recently begun to be unraveled. Here we present new insight into the fundamental role of the basic leucine zipper transcription factor NFIL3 in ILC development. We show that NFIL3 directs the development of bone marrow precursors, derived from the CLP, which give rise to all known ILC lineages including cNK cells. Additionally, we show that NFIL3 regulates the expression of the transcription factor TOX, and provide evidence that a NFIL3-TOX transcription factor cascade is central to the development of all ILC lineages.</p><p>Several transcription factors are known to play essential roles in ILC development. For example, all ILC subsets express Id2, an antagonist of E proteins that control B- and T- cell commitment (<xref ref-type="bibr" rid="bib18">Kee, 2009</xref>; <xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>). Deletion of <italic>Id2</italic> in mice abrogates the development of multiple ILC lineages (<xref ref-type="bibr" rid="bib53">Yokota et al., 1999</xref>; <xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>), although NK developmental defects arise only during NK maturation (<xref ref-type="bibr" rid="bib2">Boos et al., 2007</xref>). The HMG factor TOX is required for the development of cNK cells and ILC3 (<xref ref-type="bibr" rid="bib1">Aliahmad et al., 2010</xref>), and the transcription factor PLZF is required for the differentiation of non-NK cell ILC subsets (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>). Most recently, the basic leucine zipper transcription factor NFIL3 was found to be essential for the development of all known ILC lineages, including cNK cells (<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>; <xref ref-type="bibr" rid="bib40">Seillet et al., 2014a</xref>).</p><p>Lineage tracing studies with <italic>Id2</italic> and <italic>Zbtb16</italic> (encoding PLZF) reporter mice have identified two distinct progenitor populations that develop into multiple ILC lineages but not cNK cells (<xref ref-type="bibr" rid="bib14">Hoyler et al., 2012</xref>; <xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>; <xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>). The PLZF-dependent ILCP differentiates into non-NK ILC1, ILC2, and ILC3, but not cNK cells (<xref ref-type="bibr" rid="bib3">Constantinides et al., 2014</xref>). Similarly, the common ‘helper-like’ innate lymphoid progenitor (CHILP) can differentiate into non-NK ILC1, ILC2 and NK1.1<sup>+</sup> NKp46<sup>+</sup> ILC3 but not cNK cells (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>). This suggests that the ILCP and the CHILP may lie downstream of a common ILC progenitor that gives rise to all ILC lineages including cNK cells.</p><p>We have provided evidence that NFIL3 is required for the differentiation of a multipotent ILC precursor population, the αLP, from the CLP. αLP gave rise to all ILC lineages including cNK cells. Although αLP lack B cell differentiation potential, they retain some residual T cell differentiation potential (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>). However, the CXCR6<sup>+</sup> αLP subpopulation, which accounted for ∼4% of αLP cells in adult bone marrow, differentiated into all ILC lineages including cNK cells, but not B- or T-cells. This suggests that CXCR6<sup>+</sup> αLP represent committed ILC precursors that give rise to all ILC lineages including cNK cells. Thus, these cells are likely to lie developmentally upstream of the described ILCP and CHILP populations.</p><p>Our findings suggest that NFIL3 activation of TOX expression is a key mechanism by which NFIL3 directs ILC development. NFIL3 is required for <italic>Tox</italic> expression in the CLP, and directs <italic>Tox</italic> expression through direct binding to the <italic>Tox</italic> promoter. Since TOX has been shown to direct the development of multiple ILC lineages, including cNK cells and ILC3 (<xref ref-type="bibr" rid="bib1">Aliahmad et al., 2010</xref>), this suggests that activation of TOX expression is a key mechanism by which NFIL3 influences ILC development. This idea is supported by our finding that forced <italic>Tox</italic> expression in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> bone marrow progenitors rescues the ILC developmental defect and restores differentiation of cNK, non-NK ILC1, ILC2, and ILC3. Together, these data support the idea that a NFIL3-TOX transcription factor cascade plays a fundamental role in the development of all ILC lineages. Recent studies have shown that forced expression of <italic>Eomes</italic> can rescue cNK cell development from <italic>Nfil3</italic><sup><italic>−/−</italic></sup> hematopoietic progenitors (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>). However, because <italic>Eomes</italic> is not expressed in CLPs (<xref ref-type="fig" rid="fig5">Figure 5A</xref>; <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>) and <italic>Eomes</italic> deficiency only impacts cNK cells but no other ILCs (<xref ref-type="bibr" rid="bib20">Klose et al., 2014</xref>), <italic>Eomes</italic> is unlikely to mediate the NFIL3-dependent development of non-NK ILCs, and may lie developmentally downstream of NFIL3-TOX during NK cell development.</p><p>Although NFIL3 is required for the development of the major ILC types and their precursors, some ILC subtypes appear to be NFIL3-independent. For example, certain NK cells, including salivary gland NK cells (<xref ref-type="bibr" rid="bib4">Cortez et al., 2014</xref>) and tissue-resident NK cells (<xref ref-type="bibr" rid="bib43">Sojka et al., 2014</xref>), are not impacted by <italic>Nfil3</italic> deficiency, in contrast to conventional NK cells. Extramedullary development of thymic NK cells is also independent of NFIL3 (<xref ref-type="bibr" rid="bib5">Crotta et al., 2014</xref>). In addition, during mouse cytomegalovirus infection, NK cells in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice expand to numbers similar to those in wild-type mice through an IL-15-dependent mechanism (<xref ref-type="bibr" rid="bib7">Firth et al., 2013</xref>). This suggests that the requirement for NFIL3 can be overridden by cytokine signaling during infection. Finally, despite the strict requirement for NFIL3 in bone marrow ILC precursor development (αLP and CHILP), <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice appear to have normal lymph nodes (<xref ref-type="bibr" rid="bib47">Spits and Di Santo, 2011</xref>; <xref ref-type="bibr" rid="bib41">Seillet et al., 2014b</xref>) and only moderately impaired Peyer's patch development (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>)(<xref ref-type="bibr" rid="bib10">Geiger et al., 2014</xref>). One possibility is that fetal LTi cell function may be preserved in the absence of <italic>Nfil3</italic>, which could account for the presence of lymph nodes in <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice.</p><p><italic>Nfil3</italic> is regulated by the circadian clock and thus its expression varies diurnally in multiple tissues and cells (<xref ref-type="bibr" rid="bib5a">Duez et al., 2008</xref>; <xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). We previously showed that <italic>Nfil3</italic> expression varies diurnally in T cells and that NFIL3 synchronizes T<sub>H</sub>17 lineage specification to the day-night light cycle (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). Synchronization is essential for T<sub>H</sub>17 cell homeostasis, as circadian disruption by chronic light cycle perturbation elevates intestinal T<sub>H</sub>17 cell frequencies and increases susceptibility to intestinal inflammation (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). The finding that NFIL3 is required for the development of committed ILC precursors suggests that precursor differentiation may also be synchronized with diurnal light cycles through a similar mechanism. Future studies will examine whether <italic>Nfil3</italic> expression is diurnally regulated in the CLP, whether precursor generation is synchronized to circadian light cycles in an NFIL3-dependent manner, and whether disruption of circadian light cycles leads to dysregulated ILC development.</p><p>Altogether, our findings provide new insight into the defining role of NFIL3 in ILC development. Identification of a committed pan-ILC precursor should allow further insight into the developmental pathways that drive ILC cell fate decisions. Because of the general importance of ILCs in immune defense, NFIL3-dependent pathways may provide new targets for treatment of inflammatory and infectious diseases.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Mice</title><p><italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice were obtained from Dr. Paul B. Rothman (Johns Hopkins University) (<xref ref-type="bibr" rid="bib17">Kashiwada et al., 2010</xref>), and were maintained by heterozygous breeding in the Specific Pathogen Free (SPF) mouse facility at the University of Texas Southwestern Medical Center at Dallas. <italic>Rag1</italic><sup><italic>−/−</italic></sup> mice (B6.129S7-Rag1<sup>tm1Mom</sup>/J), CD90.1<sup>+</sup> mice (B6.PL-<italic>Thy1</italic><sup><italic>a</italic></sup>/CyJ), CD45.1<sup>+</sup> mice (B6.SJL-<italic>Ptprc</italic><sup><italic>a</italic></sup> <italic>Pepc</italic><sup><italic>b</italic></sup>/BoyJ), Id2-eGFP reporter mice (B6.129S(Cg)-<italic>Id2</italic><sup><italic>tm2.1Blh</italic></sup>/ZhuJ), and RORγt-GFP reporter mice (B6.129P2(Cg)-<italic>Rorc</italic><sup><italic>tm2Litt</italic></sup>/J) were purchased from the Jackson Laboratory, Bar Harbor, Maine. <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice were intercrossed with <italic>Rag1</italic><sup><italic>−/−</italic></sup> mice to create <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> double knockout mice. <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> mice (B10;B6-<italic>Rag2</italic><sup><italic>tm1Fwa</italic></sup> <italic>II2rg</italic><sup><italic>tm1Wjl</italic></sup>) were purchased from Taconic Farms, New York. All procedures described in this study were performed in accordance with protocols approved by the Institutional Animal Care and Use Committees (IACUC) of the UT Southwestern Medical Center.</p></sec><sec id="s4-2"><title>Isolation and analysis of intestinal lamina propria lymphocytes</title><p>Lamina propria lymphocytes (LPLs) were isolated from the intestine as previously described (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). Briefly, intestines were dissected from mice and Peyer's patches were removed. Intestines were cut into small pieces and thoroughly washed with ice-cold PBS. Epithelial cells were removed by incubating intestinal tissues in Hank's buffered salt solution (HBSS) supplemented with EDTA and DTT, followed by extensive washing with PBS. Residual tissues were digested by Collagenase IV (Sigma, St. Louis, Missouri), DNase I (Sigma) and Dispase (BD Biosciences, San Jose, California) for 1 hr at 37°C. Cells were filtered through 100 μm cell strainers and applied onto a 40%:80% Percoll gradient (GE Healthcare, Pittsburgh, Pennsylvania), in which lamina propria lymphocytes were found at the interface of 40% and 80% fractions.</p><p>Livers were dissected from mice and cut into small pieces, followed by digestion with Collagenase IV (Sigma), DNase I (Sigma) and Dispase (BD Biosciences) for 1 hr at 37°C. Residual tissues were forced through 100 μm cell strainers. Cells were spun down and applied onto a 40%:80% Percoll gradient as for the LPLs.</p><p>Isolated lymphocytes were washed with PBS with 2 mM EDTA and 3% fetal bovine serum (FBS) and F<sub>c</sub> receptors were blocked with α-CD16/32 (2.4G2). Cells were then stained with antibodies against cell surface markers including α-CD3ε (500A2), α-CD19 (ebio1D3), α-CD5 (53-7.3), α-TCRβ (H57-597), α-TCRγδ (GL3), α-NK1.1 (PK136), α-Sca1 (D7), α-KLRG1 (2F1), α-NKp46 (29A1.4), α-CD45 (30-F11), α-CD45.1 (A20), α-CD45.2 (104), α-hCD2 (RPA-2.10), and α-CD127 (A7R34). Cells were fixed/permeabilized with eBiosciences (San Diego, California) Mouse Regulatory T Cell Staining Kit #3 per the manufacturer's instructions, and subjected to nuclear staining with α-RORγ (AFKJS-9), α-GATA3 (TWAJ), α-T-BET (4B10) and α-EOMES (Dan11mag). Cells were analyzed with an LSRII (BD Biosciences, San Jose, California) or CyAn ADP (Beckman Coulter, Jersey City, New Jersey) flow cytometer and data were processed with FlowJo software (Tree Star, Ashland, Oregon).</p></sec><sec id="s4-3"><title>Isolation and analysis of bone marrow progenitors</title><p>Femur and tibia were dissected from adult mice and bone marrow cells were released in PBS buffer containing 2 mM EDTA and 3% FBS with a mortar and pestle. Cells were filtered through 70 μm cell strainers and blocked with α-CD16/32 (2.4G2), followed by incubation with biotinylated lineage markers (Lin) antibodies including α-CD3ε (145-2C11), α-B220 (RA3-6B2), α-CD11b (M1/70), α-Gr1 (RB6-8C5), α-Erythroid Cells (TER119), α-CD5 (53-7.3), α-TCRγδ (GL3), and α-NK1.1 (PK136). Cells were then washed and incubated with α-biotin magnetic microbeads (Miltenyi Biotec, San Diego, California). Lineage-negative cells were enriched by an autoMACS sorter with the ‘Depletes’ setting. Surface staining was performed with antibodies including α-biotin (Bio3-18E7), α-CD45 (30-F11), α-cKit (2B8), α-CD127 (A7R34), α-Sca1 (D7), α-Flt3 (A2F10) and α-α<sub>4</sub>β<sub>7</sub> integrin (DATK32) and α-CXCR6 (221002). FACS sorting was performed with a FACSAria cell sorter (BD Biosciences) while flow cytometry analysis was carried out with an LSRII (BD Biosciences). In both cases, LSK cells were identified as Lin<sup>−</sup> Sca1<sup>+</sup> cKit<sup>+</sup>, CLP as Lin<sup>−</sup> cKit<sup>low</sup> CD127<sup>+</sup> Sca1<sup>low</sup> Flt3<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>−</sup>, αLP as Lin<sup>−</sup> cKit<sup>low</sup> CD127<sup>+</sup> Sca1<sup>low</sup> Flt3<sup>-</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup>, ILC2P as Lin<sup>−</sup> CD127<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD25<sup>+</sup> Sca1<sup>+</sup>, and CHILPs as Lin<sup>−</sup> CD127<sup>+</sup> α<sub>4</sub>β<sub>7</sub><sup>+</sup> CD25<sup>-</sup> Flt3<sup>-</sup>. Data were processed with FlowJo software (Tree Star).</p></sec><sec id="s4-4"><title>Cell transfer assay</title><p>αLPs were purified from CD45.1<sup>+</sup> mice by FACS sorting as described above. In order to obtain a large number (∼1000) CXCR6<sup>+</sup> αLP, femur and tibia from 20 CD45.1<sup>+</sup> mice were pooled together for the cell isolation. <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> recipient mice were sublethally irradiated with a dose of 4.2 Gy on the same day with an XRAD320 irradiator (Precision X-ray, Inc, North Branford, Connecticut). Cells were transplanted into recipient mice by retro-orbital injection. ILCs in recipient mice were examined 4–6 weeks later.</p><p>For CLP co–transfer experiments, wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> CLP cells were purified by FACS sorting and mixed at a 1:1 ratio before transplantation into sublethally irradiated <italic>Rag2</italic><sup><italic>−/−</italic></sup><italic>;Il2rg</italic><sup><italic>−/−</italic></sup> recipient mice.</p><p>For LSK co-transfer, CD90.1<sup>+</sup> recipient mice were lethally irradiated with two doses of 5 Gy on the same day. FACS-purified LSK cells from wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice were mixed at a 1:1 ratio and transplanted into recipient mice by retro-orbital injection.</p></sec><sec id="s4-5"><title>In vitro differentiation assays</title><p>αLPs were purified by FACS sorting as described above. For bulk culture, ∼25 cells were co-cultured on a monolayer of OP9 cells (OP9-GFP) or OP9 cells stably expressing the Notch ligand Delta-like 1 (OP9-DL1) in αMEM media. To induce ILC2, the culture medium was supplemented with 20 ng/ml Stem Cell Factor (SCF, PeproTech, Rocky Hill, New Jersey), 20 ng/ml IL-7 (BioLegend, San Diego, California) and 20 ng/ml IL-2 (BioLegend). To induce ILC3, 20 ng/ml SCF, IL-7 and IL-23 (BioLegend) were added to the medium. The culture medium was replaced every 3–4 days and, after 14 days, cells were stained and analyzed by flow cytometry. For clonal differentiation, OP9-DL1 cells were irradiated at 1500 rad and seeded at a density of 10,000 cells per well in a 96-well plate. On the following day, CXCR6<sup>+</sup> αLP cells were individually sorted into the wells and cultured in αMEM media supplemented with 20 ng/ml SCF and IL-7. Cells were analyzed by flow cytometry 3 weeks later. In total, 240 cells from two independent experiments were analyzed.</p></sec><sec id="s4-6"><title>CLP transcriptome analysis</title><p>CLPs from wild-type and <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice were purified as described above. Total RNA was isolated with the PicoPure RNA Isolation Kit (Life Technologies, Grand Island, New York). RNA quality and quantity were determined with a Bioanalyzer (Agilent Genomics) with a Pico chip. Samples with RNA integrity numbers (RIN) larger than 8 were subjected to further processing and hybridized to the Mouse WG-6 V2 BeadChips (Illumina, San Diego, California) by the UT Southwestern Microarray Core Facility.</p><p>Microarray images were processed and annotated with GenomeStudio (Illumina). Differential gene expression analysis was performed using R together with bioConductor and the Limma package (<xref ref-type="bibr" rid="bib42">Smyth et al., 2005</xref>; <xref ref-type="bibr" rid="bib35">Ritchie et al., 2011</xref>). Briefly, signal intensities were first log<sub>2</sub>-transformed, followed by background correction and quantile normalization with the NEQC function. Empirical reliabilities of samples were estimated by the arrayWeights function, which gave each sample a weight score accordingly. Samples were then fitted into a weighted linear model by lmFit to detect differentially expressed genes.</p></sec><sec id="s4-7"><title>Tox rescue experiment</title><p>TOX-coding or NFIL3-coding sequences (CDS) were cloned by PCR from total mouse thymus cDNA into the bicistronic retroviral vector MSCV-IRES-hCD2 (a gift from Dr Chandrashekhar Pasare at UT Southwestern) to generate a TOX-encoding plasmid, MSCV-<italic>Tox</italic>-IRES-hCD2 and an NFIL3-encoding plasmid, MSCV-<italic>Nfil3</italic>-IRES-hCD2. The MSCV-IRES-hCD2, MSCV-<italic>Tox</italic>-IRES-hCD2, and MSCV-<italic>Nfil3</italic>-IRES-hCD2 plasmids were transfected into the Plat-E packaging cell line (<xref ref-type="bibr" rid="bib27">Morita et al., 2000</xref>) with FugeneHD (Promega, Madison, Wisconsin) to produce retroviral particles. Cell culture supernatant was harvested 48 and 72 hr post transfection. Cell debris was first cleared by spinning at 400×<italic>g</italic> for 10 min, followed by passage through 0.2 μm sterile filters.</p><p>LSK cells were purified from <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice by FACS sorting as described above and seeded into round-bottom 96-well plates at a density of 10,000 cells/well in STEMSPAN Serum-Free Expansion Medium (SFEM) (Stemcell Technologies, Vancouver, Canada) (<xref ref-type="bibr" rid="bib57">Zheng et al., 2012</xref>). During retroviral transduction, cells were mixed with an equal volume of cleared retrovirus-containing cell culture supernatant, supplemented with 2 U/ml Heparin (Sigma), 10 ng/ml mouse Stem Cell Factor (SCF, Peprotech), 20 ng/ml mouse Thrombopoietin (TPO, Peprotech), 10 ng/ml mouse Fibroblast Growth Factor (FGF-1, Life Technologies) and 4 μg/ml polybrene (Sigma). Spinoculation was carried out at 1200×<italic>g</italic> for 90 min at 32°C to enhance retroviral transduction. 3 hr later, cell media was replaced with fresh STEMSPAN SFEM media supplemented with the above cytokines but without polybrene. Transduction was performed on two consecutive days using retroviral supernatant harvested 48 and 72 hr post transfection, respectively.</p><p>On day 3, CD45.1<sup>+</sup> wild-type recipient mice were lethally irradiated at two doses of 5 Gy as described above. LSK cells were collected from the 96-well plate with Cell Dissociation Buffer (Life Technologies) and washed with sterile PBS. 2000–4000 cells were transferred into recipient mice in 200 μl sterile PBS by retro-orbital injection. ILCs in recipient mice were examined 5–6 weeks later.</p></sec><sec id="s4-8"><title>shRNA knockdown of NFIL3</title><p>Five independent shRNA constructs (sh38-sh42) targeting mouse NFIL3 and a control construct containing scramble sequences (pLKO.1-scramble) were purchased from Sigma. To identify shRNA constructs that could effectively knock down NFIL3, 1 μg of shRNA plasmid and 1 μg of NFIL3-encoding plasmid (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>) were co-transfected into HEK293T cells in a 6-well plate with FugeneHD (Promega). Cells were harvested 36 hr later, lysed and used for western blotting with anti-NFIL3 antibody.</p><p>Two shRNA constructs that could effectively knock down NFIL3 were identified: sh39 and sh40. These constructs as well as the pLKO.1-scramble vector were each co-transfected with the packaging plasmids pCMVDR9 and pVSVG into HEK293T cells. Cell culture supernatants were harvested 48 and 72 hr later and cleared by spinning and filtering as described above. Lentiviral particles were concentrated by ultracentrifugation at 75,000×<italic>g</italic> for 2 hr and resuspended in RPMI media.</p><p>EL4 cells were mixed with lentiviral particles in the presence of 4 μg/ml polybrene, and spinoculated at 1200×<italic>g</italic> for 90 min at 32°C. 2 days later, EL4 were selected with 8 μg/ml puromycin for 2 weeks. Live cells were sorted with a FACSAria cell sorter as they excluded propidium iodide.</p></sec><sec id="s4-9"><title>NFIL3 overexpression in EL4 cells</title><p>NFIL3 coding sequences were subcloned into MSCV-IRES-hCD2 to generate MSCV-<italic>Nfil3</italic>-IRES-hCD2. MSCV-IRES-hCD2 and MSCV-<italic>Nfil3</italic>-IRES-hCD2 were then transfected into Plat-E cells to produce retroviral particles as described above, which were then used to transduce EL4 cells. 3 days after transduction, EL4 cells were stained with anti-hCD2 and hCD2<sup>+</sup> EL4 cells were purified with a FACSAria cell sorter. Sorted cells were maintained in RPMI media for another 3–4 days, followed by staining and sorting again. The resulting cells were expanded and <italic>Nfil3</italic> expression was examined by SYBR green-based real-time PCR.</p></sec><sec id="s4-10"><title>Chromatin Immunoprecipitation (ChIP)</title><p>ChIP experiments were carried out as previously described (<xref ref-type="bibr" rid="bib56">Yu et al., 2013</xref>). Briefly, EL4 cells or NFIL3-overexpressing EL4 cells were cultured in RPMI medium at ∼0.8 × 10<sup>6</sup> cells/ml. Cells were harvested and fixed with 1% formaldehyde for 10 min in the dark, which was quenched by adding glycine to a final concentration of 0.15 M. Nuclei were released with a Dounce homogenizer (Wheaton, Millville, New Jersey) in Nuclear Isolation Solution containing 10 mM Tris pH 7.4, 5 mM MgCl<sub>2</sub>, 25 mM KCl and 250 mM sucrose, and purified by spinning at 1000×<italic>g</italic> for 10 min over Hypertonic Solution containing 10 mM Tris pH 7.4, 5 mM MgCl<sub>2</sub>, 25 mM KCl and 30% (wt/vol) sucrose. Purified nuclei were used for ChIP with the Magna ChIP assay kit (Millipore, Billerica, Massachusetts) per the manufacturer's instructions. The <italic>Tox</italic> promoter was detected by SYBR green-based real-time PCR with specific primers: Tox-ChIPF6: 5′-GACACTGACAGCAAGGACCA-3′ and Tox-ChIPR6: 5′-CAGGGCTTCATAGCACCGAT-3′, targeting nucleotide −2105 to nucleotide −1867 in the <italic>Tox</italic> promoter. Enrichment of the <italic>Tox</italic> promoter was determined by normalizing the level of the <italic>Tox</italic> promoter in DNA pulled down with an anti-NFIL3 antibody to that pulled down with an IgG control.</p></sec><sec id="s4-11"><title><italic>Tox-luciferase</italic> reporter assay</title><p>A 2.3 kb fragment (−2133 to 232) of the <italic>Tox</italic> promoter was cloned into the pGL3-Basic vector to drive firefly <italic>luciferase</italic> expression (the <italic>Tox-luciferase</italic> reporter). HEK293T cells were cultured in a 96-well plate overnight and were co-transfected with the <italic>Tox-luciferase</italic> reporter and an empty or NFIL3-encoding vector. A pCMV-Renilla-Luciferase reporter was co-transfected into HEK293T cells to serve as an internal control. Luciferase activities were detected using the Dual-Glo Luciferase Assay kit (Promega) and measured with a SpectraMax M5e plate reader (Molecular Devices, Sunnyvale, California). Firefly luciferase activities in each sample were first normalized against Renilla luciferase activities in the same sample and then normalized against that in cells transfected with the empty vector.</p></sec><sec id="s4-12"><title>Citrobacter rodentium infection</title><p>The <italic>C. rodentium</italic> (DBS100) strain was originally obtained from ATCC (Manassas, Virginia). To infect mice, <italic>C. rodentium</italic> (DBS100) was first inoculated into Luria-Bertani <bold>(</bold>LB<bold>)</bold> broth overnight at 37°C with shaking in the presence of 50 μg/ml nalidixic acid, and was subcultured into fresh LB media the next morning until OD<sub>600</sub> = ∼0.8–1.0. Bacteria were then harvested by centrifugation and resuspended in sterile PBS. <italic>Rag1</italic><sup><italic>−/−</italic></sup> and <italic>Nfil3</italic><sup><italic>−/−</italic></sup>;<italic>Rag1</italic><sup><italic>−/−</italic></sup> mice were deprived of food the night before infection and were orally gavaged with 5 × 10<sup>9</sup> CFU in 200 μl sterile PBS. The number of viable <italic>C. rodentium</italic> (DBS100) in the inoculum was confirmed by retrospective plating on nalidixic acid-containing LB-agar plates. Mouse disease conditions were monitored by weight loss.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Cassie Behrendt Boyd and Tess Leal for assistance with mouse experiments. We thank Dr Nicolai Van Oers and Ashley Hoover for sharing mouse thymus total cDNA, and Dr Hua Wang for assistance with cell irradiation. We also thank Dr Juan Carlos Zúñiga-Pflücker for providing us with the OP9 cell lines, and Dr Paul Rothman for providing the <italic>Nfil3</italic><sup><italic>−/−</italic></sup> mice. This work was supported by NIH R01 DK070855 (LVH), a Burroughs Wellcome Foundation New Investigators in the Pathogenesis of Infectious Diseases Award (LVH), and the Howard Hughes Medical Institute (LVH).</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>XY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>YW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>CCZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>MD, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>YL, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>KAR, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>LVH, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: All animal experiments were approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center, and the approved animal protocol number is 1004-06-04-1. The institutional guidelines for the care and use of laboratory animals were followed.</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><sec sec-type="datasets"><title>Major dataset</title><p>The following dataset was generated:</p><p><related-object content-type="generated-dataset" source-id="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62337" source-id-type="uri" id="dataro1"><collab collab-type="author">Yu X</collab>, <collab collab-type="author">Wang Y</collab>, <collab collab-type="author">Deng M</collab>, <collab collab-type="author">Li Y</collab>, <collab collab-type="author">Ruhn KA</collab>, <collab collab-type="author">Zhang CC</collab>, <collab collab-type="author">Hooper LV</collab>, <year>2014</year><x>, </x><source>The basic leucine zipper transcription factor NFIL3 directs the development of a common innate lymphoid cell precursor</source><x>, </x><object-id pub-id-type="art-access-id">GSE62337</object-id><x>; 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the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “NFIL3 directs the development of a common innate lymphoid cell precursor” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Tadatsugu Taniguchi (Senior editor), a Reviewing editor, and 3 reviewers.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The manuscript presents a substantial body of data supporting the idea that the transcription factor NFIL3 is crucial for the entire innate lymphoid cell (ILC) lineage commitment by showing, inter alia, that NFIL3 deficiency results in a strong reduction of the 'alpha-LP' lymphoid progenitor (aLP) cells that contain precursors for cNK and ILC3 cells, as well as a loss of the putative downstream common 'helper-like' innate progenitor (CHILP) cells. Further, the manuscript significantly extends earlier observations by showing that the ILC progenitor potential in aLP compartment is likely to be contained in the previously identified CXCR6-expressing sub-population. Finally, using a transcriptome survey, the manuscript generates the hypothesis that Tox is a downstream target of Nfil3 relevant for ILC development, and tests it with Tox-specific siRNA and overexpression approaches to correlate Nfil3 expression with Tox, to show that TOX is a downstream target of NFIL3, and to demonstrate that it can rescue ILC development of NFIL3-/- precursors.</p><p>While other groups have recently published similar findings, this manuscript makes important extensions in the cellular-molecular understanding of ILC development. The data are of high quality and rapid publication is warranted. However, some issues of concern as detailed below should be resolved prior to publication.</p><p>1) Since Nfil3-deficient mice appear to have lymph nodes (e.g., <xref ref-type="bibr" rid="bib47">Spits and Di Santo, 2011</xref>) and Peyer's patches albeit fewer and smaller (present manuscript), it is arguable that LTi cell function is largely preserved during the fetal period in them. Also, NKp46- ILC3 are only mildly reduced in Nfil3-deficient mice (<xref ref-type="fig" rid="fig1">Figure 1</xref>). On the other hand, the authors show convincing data for the deficiency of the CHILP compartment, shown to be a common progenitor for helper-like ILCs including LTi cells, in the absence of Nfil3. Is it possible, for example, that Nfil3 is required for the maintenance of ILC progenitors in adult mice but not as strictly required for ILC fate decisions in the fetus? There is also literature suggesting Nfil3-indepedent generation of some ILC subsets; salivary gland NK cells (<xref ref-type="bibr" rid="bib4">Cortez et al., 2014</xref>), cNK cells after MCMV infection (<xref ref-type="bibr" rid="bib7">Firth et al., 2013</xref>), as also the mild reduction in NKP cells in Nfil3-deficient mice (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>). The discussion would benefit from acknowledgment and attempted resolution of these issues.</p><p>2) A fair proportion of the molecular connections between Nfil3 and Tox have been derived from studies with the EL4 lymphoma cell line. While it is appreciated that performing many of these analyses in CLP cells is not feasible, the manuscript needs to acknowledge that EL4 cells are relatively unrelated to any of the cell types being considered and therefore there may be caveats for the interpretations based on data from them.</p><p>3) In <xref ref-type="fig" rid="fig4">Figure 4</xref>, it is unclear how the authors excluded contamination of the aLP by CHILP, which might skew the obtained results.</p><p>4) The authors refer to literature (<xref ref-type="bibr" rid="bib24">Male et al., 2014</xref>) for the impact of NFIL3 on NKP cells, but that study found only a small reduction of NKP cells in Nfil3-deficient mice. It would therefore be helpful if the authors could present data for NKP cells in Nfil3-deficient mice in their hands.</p><p>5) <xref ref-type="fig" rid="fig2">Figure 2A</xref> shows that Nfil3 deficiency results in a 90% reduction in total aLP cells. Nonetheless, T and B cell development is not affected. It would be very useful to show if the CXCR6+ aLP subset specifically and substantially depleted in Nfil3-deficient mice, or if depletion occurs in the CXCR6- aLP cells as well.</p><p>6) In <xref ref-type="fig" rid="fig5">Figure 5F</xref>, the ILC profile from the Nfil3-deficient CD45.2+ hCD2- donor cells (without TOX overexpression) would be an important and interesting control.</p><p>7) Since the authors later show that only the CXCR6+ subset of aLP have the potential to give rise to all ILC lineages, it would be helpful if they could show the ILC transcription factor profile of these cells. Do they express any Id2? Do they express RORgt, since <xref ref-type="bibr" rid="bib31">Possot et al. (2011)</xref> showed that 40% of the CXCR6+ aLP in fetal liver were RORgt+?</p><p>8) The developmental rescue of NFIL3-/- precursors by TOX is of interest, but its quantitative significance would be best interpreted in comparison with the efficiency of rescue of these Nfil3-/- precursors with Nfil3 itself.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04406.017</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Since Nfil3-deficient mice appear to have lymph nodes (e.g.,</italic> <xref ref-type="bibr" rid="bib47"><italic>Spits and Di Santo, 2011</italic></xref><italic>) and Peyer's patches albeit fewer and smaller (present manuscript), it is arguable that LTi cell function is largely preserved during the fetal period in them. Also, NKp46- ILC3 are only mildly reduced in Nfil3-deficient mice (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref><italic>). On the other hand, the authors show convincing data for the deficiency of the CHILP compartment, shown to be a common progenitor for helper-like ILCs including LTi cells, in the absence of Nfil3. Is it possible, for example, that Nfil3 is required for the maintenance of ILC progenitors in adult mice but not as strictly required for ILC fate decisions in the fetus? There is also literature suggesting Nfil3-indepedent generation of some ILC subsets; salivary gland NK cells (</italic><xref ref-type="bibr" rid="bib4"><italic>Cortez et al., 2014</italic></xref><italic>), cNK cells after MCMV infection (</italic><xref ref-type="bibr" rid="bib7"><italic>Firth et al., 2013</italic></xref><italic>), as also the mild reduction in NKP cells in Nfil3-deficient mice (</italic><xref ref-type="bibr" rid="bib24"><italic>Male et al., 2014</italic></xref><italic>). The discussion would benefit from acknowledgment and attempted resolution of these issues</italic>.</p><p>We thank the reviewers for raising this issue and offering insightful suggestions for amplifying our discussion. We have added an additional paragraph to the Discussion section that deals with these points.</p><p><italic>2) A fair proportion of the molecular connections between Nfil3 and Tox have been derived from studies with the EL4 lymphoma cell line. While it is appreciated that performing many of these analyses in CLP cells is not feasible, the manuscript needs to acknowledge that EL4 cells are relatively unrelated to any of the cell types being considered and therefore there may be caveats for the interpretations based on data from them</italic>.</p><p>We agree that it is important to discuss this point and have now added the following sentences to the Results section:</p><p>“EL4 cells are derived from T lymphocytes, a CLP-derived lineage, and thus we cannot exclude the possibility that the regulatory relationship between <italic>Nfil3</italic> and <italic>Tox</italic> differs between T lymphocytes and CLPs. Nevertheless, our studies on CLPs and EL4 cells both support the idea that NFIL3 is an activator of <italic>Tox</italic> expression.”</p><p><italic>3) In</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref><italic>, it is unclear how the authors excluded contamination of the aLP by CHILP, which might skew the obtained results</italic>.</p><p>We cannot exclude contamination of the total αLP population by CHILP, and in fact, it is likely that CHILP cells are present in the αLP population. However, because CHILP do not give rise to cNK cells (Klose et al., <italic>Cell</italic> vol. 157, p. 340 [2014]), our data identify a distinct CXCR6<sup>+</sup> αLP committed precursor that gives rise to all four ILC lineages including cNK cells (revised <xref ref-type="fig" rid="fig4">Figure 4D</xref>). This precursor must therefore lie developmentally upstream of the CHILP. Additionally, in support of our <italic>in vitro</italic> findings, we now include data (new <xref ref-type="fig" rid="fig4">Figure 4E</xref>) showing that the CXCR6<sup>+</sup> αLP cells also give rise to all four ILC lineages (including cNK cells) <italic>in vivo</italic>.</p><p><italic>4) The authors refer to literature (</italic><xref ref-type="bibr" rid="bib24"><italic>Male et al., 2014</italic></xref><italic>) for the impact of NFIL3 on NKP cells, but that study found only a small reduction of NKP cells in Nfil3-deficient mice. It would therefore be helpful if the authors could present data for NKP cells in Nfil3-deficient mice in their hands</italic>.</p><p><xref ref-type="fig" rid="fig1">Figure 1B</xref> of <xref ref-type="bibr" rid="bib24">Male et al. (2014)</xref> actually shows a marked decline in NKP frequencies and absolute numbers in <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice. We note that the data are shown in log scale, compressing the visual differences. The percentages of both preNKP and rNKP cells among Lin- bone marrow cells declines by ∼8-10-fold in <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice. Likewise, there is an approximately 10-fold decline in the absolute numbers of both preNKP and rNKP cells in <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice. Therefore, the Male et al. paper strongly supports the idea that NFIL3 is essential for the differentiation of NKP cells.</p><p><italic>5)</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2A</italic></xref> <italic>shows that Nfil3 deficiency results in a 90% reduction in total aLP cells. Nonetheless, T and B cell development is not affected. It would be very useful to show if the CXCR6+ aLP subset specifically and substantially depleted in Nfil3-deficient mice, or if depletion occurs in the CXCR6- aLP cells as well</italic>.</p><p>We have now included an analysis of CXCR6<sup>+</sup> αLP cells in wild-type and <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice. The percentages of CXCR6<sup>+</sup> cells among αLP are comparable between wild-type and <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice (<xref ref-type="fig" rid="fig4">Figure 4A</xref>; <xref ref-type="fig" rid="fig4s1">Figure 4-figure supplement 1</xref>). As a result, the number of CXCR6<sup>+</sup> αLP cells is markedly diminished in <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice (<xref ref-type="fig" rid="fig4">Figure 4B</xref>) in parallel with the decrease in total αLP cells (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). These data support the idea that CXCR6<sup>+</sup>αLPs require NFIL3 for their development.</p><p>Since αLP cells do not give rise to B cells (<xref ref-type="fig" rid="fig3">Figure 3D</xref>), it is not surprising that B cell development is not impaired in Nfil3-/- mice despite the marked αLP deficiency. Though αLP cells retain some T cell differentiation potential (<xref ref-type="fig" rid="fig3">Figure 3D</xref> and <xref ref-type="fig" rid="fig4">Figure 4C</xref>), it is not clear to what extent αLP-derived T cells contribute to the total T cell pool in mice. There is evidence that LSK cells, which are present in normal numbers in Nfil3-/- mice, can enter the circulation and seed the thymus to initiate T cell development (Reviewed in Bhandoola and Sambandam, From stem cell to T cell: one route or many? Nat Rev Immunol. vol.6(2), p. 117-26[2006].) Thus, the marked αLP deficiency in <italic>Nfil3</italic><sup><italic>-/-</italic></sup> mice is consistent with the relatively normal T cell numbers in these mice.</p><p><italic>6) In</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5F</italic></xref><italic>, the ILC profile from the Nfil3-deficient CD45.2+ hCD2- donor cells (without TOX overexpression) would be an important and interesting control</italic>.</p><p>The TOX encoding plasmid was delivered into LSK cells by retroviral transduction/infection. It is well-documented that retroviral infection of bone marrow progenitors can by itself impact their ability to differentiate (reviewed in Banerjee et al., Hematopoietic stem cells and retroviral infection. <italic>Retrovirology</italic> vol. 7, p. 8-17 [2010]). Therefore, comparison of hCD2<sup>+</sup> and hCD2<sup>-</sup> donor cells will not provide interpretable information about the impact of TOX expression on the differentiation potential of the cells. For this reason, we have restricted our analysis to a comparison between <italic>Tox</italic>-expressing hCD2<sup>+</sup> donor cells and vector-only hCD2<sup>+</sup> donor cells.</p><p><italic>7) Since the authors later show that only the CXCR6+ subset of aLP have the potential to give rise to all ILC lineages, it would be helpful if they could show the ILC transcription factor profile of these cells. Do they express any Id2? Do they express RORgt, since</italic> <xref ref-type="bibr" rid="bib31"><italic>Possot et al. (2011)</italic></xref> <italic>showed that 40% of the CXCR6+ aLP in fetal liver were RORgt+?</italic></p><p>This is a great suggestion. However, the number of CXCR6<sup>+</sup> αLP cells in adult mouse bone marrow is exceptionally small, perhaps due to the fact that these cells represent a transitional stage between αLP and CXCR6<sup>+</sup> α4β7<sup>-</sup> cells that lie downstream of αLP (<xref ref-type="bibr" rid="bib31">Possot et al., 2011</xref>). The small number of cells makes such an analysis exceptionally challenging. As shown in revised <xref ref-type="fig" rid="fig4">Figure 4B</xref>, we detect ∼5 CXCR6<sup>+</sup> αLP cells per 3.3 million Lineage-negative bone marrow cells. As there are, on average, approximately 12 million Lineage-negative bone marrow cells per femur/tibia, this equates to ∼40 CXCR6<sup>+</sup> αLP cells per femur/tibia. The rarity of these cells makes it extremely difficult to accurately assess transcription factor expression across the population in adult mouse bone marrow by flow cytometry. The analysis in <xref ref-type="bibr" rid="bib31">Possot et al. (2011)</xref> was performed on fetal liver CXCR6<sup>+</sup> αLP cells, which account for roughly 0.15% of Linage-negative fetal liver cells and are thus much more abundant than in adult bone marrow. Finally, Possot et al. used quantitative RT-PCR to show that <italic>Rorc</italic> (encoding RORγt) expression is undetectable in CXCR6<sup>+</sup> cells from adult bone marrow.</p><p><italic>8) The developmental rescue of NFIL3-/- precursors by TOX is of interest, but its quantitative significance would be best interpreted in comparison with the efficiency of rescue of these Nfil3-/- precursors with Nfil3 itself</italic>.</p><p>This is a great suggestion and we have now included data showing the efficiency of developmental rescue of <italic>Nfil3</italic><sup><italic>-/-</italic></sup> precursors with NFIL3 in <xref ref-type="fig" rid="fig5">Figure 5F</xref>. The results show that developmental rescue is comparable between TOX and NFIL3, with no statistically-significant differences between the two across the various ILC subsets. Although the average rescue efficiency for the ILC2 subset trended higher with NFIL3, the overall difference was not statistically significant.</p></body></sub-article></article> |