The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development

We previously reported that Bcl11b is expressed in all T cells, from DN2 thymocytes to mature T cells, using the Bcl11b-Tdtomato reporter mouse (Bcl11b^Td/Td; Li et al., 2010b). The Bcl11b gene expression was recently detected in ILC2 in RT-PCR (Wong et al., 2012; Yang et al., 2013). We took advantage of the Bcl11b^Td/Td reporter mouse and further systematically explored Bcl11b expression in ILC development. At steady state, Bcl11b expression was found in all ILC2s in the BM, the lung, the mesenteric LN (MLN), and small intestine lamina propria (siLP; Fig. 1 A and Fig. S1, A–C and G). In contrast, ILC1s and ILC3s, including intraepithelial lymphocyte (IEL) ILC1s, liver DX5⁺ and DX5⁻ NK cells, spleen NK cells, lamina propria lymphocyte (LPL) NCR⁺ILC3s, LPL CD4⁺ or CD4⁻ lymphoid tissue inducers (LTi’s), or LPL NKp46⁺NK1.1⁺ cells (including conventional NK cells, ILC1s, and ILC3s; Klose et al., 2014), did not have detectable Bcl11b expression (Fig. 1 A and Fig. S1, D–G).

We next examined Bcl11b expression in ILC2s in the presence of IL-25 and IL-33, as recent studies suggest that ILC2s are elicited by these cytokines (for example, Neill et al., 2010). To minimize the background of the adaptive immune responses and to eliminate potential contamination with T cells, we crossed Bcl11b^Td/Td reporter mouse to the Rag1^−/− knockout mouse to obtain Bcl11b^Td/TdRag1^−/− mice. In Bcl11b^Td/TdRag1^−/− mice, 0.85% of the lung leukocytes expressed Bcl11b, and 20% of them expressed the mature ILC2 marker KLRG1 (Fig. 1 B; Hoyler et al., 2012). Once IL-25 or IL-33 was administered, Bcl11b-expressing lung leukocytes increased to 34% and 36%, respectively (Fig. 1 B). Notably, almost all of the expanded cells expressed KLRG1 (Fig. 1 B).

Similar to Th2 cells, ILC2s secrete type 2 cytokines such as IL-5 and IL-13 (Spits et al., 2013). We next purified Lin⁻Bcl11b⁺ leukocytes from the BM and the lung of Bcl11b^Td/TdRag1^−/− mice and briefly stimulated these cells ex vivo with PMA and Ionomycin for IL-5 production (Hoyler et al., 2012). Many Lin⁻Bcl11b⁺ cells produced IL-5 (Fig. 1 C). In contrast, the Lin⁻Bcl11b⁻ cells from both the BM and the lung did not have detectable IL-5 production (Fig. 1 C). These results thus demonstrate that Bcl11b is specifically expressed in ILC2s in ILCs.

Recent studies indicate that ILCs are differentiated from progenitors, which, in turn, originate from CLPs in the BM (Spits et al., 2013; Constantinides et al., 2014; Klose et al., 2014). Analysis of reporter mouse lines has identified two overlapping ILCP populations: common helper innate lymphoid progenitor (ChILP) cells that express ID2 (Klose et al., 2014) and ILCPs, which are characterized by the expression of promyelocytic leukemia zinc finger (Constantinides et al., 2014). Both progenitor populations can give rise to three types of ILCs but are heterogeneous as revealed in in vitro single-cell developmental fate assays. Notably, ∼28% of cells in the ChILP or ILCP compartment were reported to generate only ILC2s in vitro (Constantinides et al., 2014; Klose et al., 2014). High levels of Bcl11b expression mark the commitment of the T cell lineage in the thymus (Li et al., 2010a,b; Yu et al., 2012). We asked whether Bcl11b is also involved in early development of ILC2s. Consistent with previous studies, Bcl11b expression was not detected in either the Lin⁻Sca1⁺c-Kit⁺ (LSK) compartment or CLP (Fig. 2 A; Li et al., 2010b). However, in the ChILP compartment defined by Lin⁻Flt3⁻IL-7Rα⁺α₄β₇⁺CD27⁺CD25⁻CD244⁺ (Fig. S2), coincidentally, Bcl11b expression was found in ∼28% of ChILPs (Fig. 2 A). Expression of Bcl11b appeared to increase from Bcl11b⁺ChILPs to ILC2 progenitors (ILC2Ps; Fig. 2 B and Fig. S2). Moreover, Bcl11b⁺ChILPs expressed other ILC2 genes such as Gata3 and ICOS, but little IL-33R (Fig. 2 C). To test whether these Bcl11b-expressing ChILPs overlap with those ones that generate only ILC2s (Constantinides et al., 2014; Klose et al., 2014), we intravenously injected purified Bcl11b⁺ChILPs into sublethally irradiated alymphoid Rag2^−/−Il2rg^−/− mice to examine their in vivo developmental potential. 6–8 wk after transplantation, siLP cells from recipients were harvested for ILC analysis. Bcl11b⁺ChILPs produced only ILC2s, similar to what ILC2Ps (Lin⁻Flt3⁻IL-7Rα⁺α₄β₇⁺CD27⁺CD25⁺CD244⁻) did (Fig. 2 D; Hoyler et al., 2012; Klose et al., 2014). On the other hand, both CLPs and Bcl11b⁻ChILPs produced CD4⁺ and CD4⁻ LTi cells besides ILC2s (Fig. 2 D). These results indicate that these Bcl11b⁺ChILPs are already committed to the ILC2 lineage and may represent an earlier progenitor population (CD25⁻IL-33R⁻) than ILC2P (CD25⁺IL-33R⁺). We subsequently performed short-term in vitro fate assay, which again demonstrated that Bcl11b⁺ChILPs only produced ILC2 (Fig. 2 E). Therefore, Bcl11b expression in ChILPs marks early committed ILC2Ps.

We started investigating Bcl11b functions in ILC2 development by examining the Bcl11b heterozygous germline loss of function mutant mice (+/−) because the homozygous mutants die at birth (Wakabayashi et al., 2003). Compared with the wild-type control mice (+/+), the heterozygotes had ∼59% of ILC2s (Lin⁻IL-33R⁺IL-7Rα⁺CD25⁺) in the BM (Fig. 3 A and Fig. S3 A), indicating that Bcl11b has a dose-dependent role in ILC2 development. Surprisingly, compared with the wild-type control mice, the heterozygotes had significantly higher percentages of mature ILC2s in the BM defined by CD25⁺KLRG1⁺ (Fig. 3 B and Fig. S3 A), indicating that Bcl11b might also normally prevent production of mature ILC2s from progenitors. On the other hand, ILC2 numbers in the lung of the heterozygotes were not significantly reduced (Fig. 3 C and Fig. S3 B).

We performed further analysis of Bcl11b in ILC2 development using the Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} conditional knockout mice (Li et al., 2010b). These mice were grossly normal and fertile as previously reported (Li et al., 2010b). Bcl11b deletion in these mice was achieved by administration of Tamoxifen (Tam; the treated mice are referred to as Bcl11b^fl/fl). We chose Bcl11b^+/+Rosa26^{CreERT2/CreERT2} mice as the control (the treated mice are referred to as Bcl11b^+/+) to help exclude the potential defects from Cre toxicity. We analyzed the mice 2–3 wk after Tam administration. Bcl11b deletion caused severe phenotypes in the BM ILC2 compartment, with only ∼6% left compared with the control (Fig. 4, A and B). In contrast, lung ILC2s were not obviously reduced (Fig. 4 C and Fig. S4 A). Rather, more of them expressed KLRG1 (Fig. 4 D and Fig. S4 A), which is consistent with the result that the Bcl11b heterozygous mutants have increased mature ILC2s (Fig. 3 B and Fig. S3 A). This result shows that Bcl11b inactivation causes severe defects in early development of ILC2s in the BM.

To address whether the defects in ILC2s caused by Bcl11b deficiency were cell autonomous or intrinsic to hematopoietic cells, we intravenously injected Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} or Bcl11b^+/+Rosa26^{CreERT2/CreERT2} BM cells into lethally irradiated CD45.1⁺ C57B6 recipients. The recipients were allowed 6–8 wk for donor cell reconstitution before Tam was administered to delete Bcl11b. BM cells from these mice were subsequently harvested 3–4 wk after Tam treatment. Again, only ∼6% of donor ILC2s was left in the BM after Bcl11b deletion (Fig. 4 E and Fig. S4 B). In contrast, no obvious changes were found in BM ILC1Ps (Lin⁻NK1.1⁺NKp46⁺IL7Rα⁺CD49a⁺; Fig. 4 F and Fig. S4 C; Klose et al., 2014).

We next purified BM hematopoietic progenitors (LSKs) from either Tam-treated Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} (Bcl11b^fl/fl) or control mice and intravenously injected these cells into lethally irradiated alymphoid Rag2^−/−Il2rg^−/− recipients (CD45.1⁺; Serafini et al., 2014) with helper BM cells (CD45.1⁺). After 8 wk of engraftment, the recipients were analyzed for ILC engraftment. Control LSKs (Bcl11b^+/+) efficiently generated ILC2s in the BM, the lung, and the siLP, as well as ILC1Ps in the BM (Fig. 4, G–K, and Fig. S5, A–C). In contrast, Bcl11b^fl/fl LSKs generated no ILC2s in the BM of chimeras (Fig. 4, G and H), and there were barely any detectable ILC2s in the lung or in the siLP (Fig. 4, J and K, and Fig. S5, B and C). On the other hand, ILC1Ps in the BM or LPL CD4⁺ or CD4⁻ LTi cells were not affected by Bcl11b deletion (Fig. 4 I and Fig. S5, A and C).

It was reported that the OP9-DL1 culture system allows CLPs to differentiate to ILC2s (Wong et al., 2012). We purified Bcl11b^fl/fl CLPs and co-cultured them with OP9-DL1 stromal cells. No ILC2s were produced from Bcl11b^fl/fl CLPs (Fig. 5 A), which is consistent with the previous in vivo data. Our previous study demonstrates that Bcl11b is essential for T cell identity maintenance (Li et al., 2010b). To investigate the functions of Bcl11b in the committed ILC2s and to test whether Bcl11b is also required for ILC2 identity maintenance, we purified ILC2Ps from Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} conditional knockout mice and cultured them on OP9-DL1 stromal cells to produce ILC2s. We subsequently treated the cultured cells with Tam to delete Bcl11b. After IL-25 or IL-33 stimulation, Bcl11b^fl/fl ILC2s were still able to produce IL-5 but not IFN-γ (Fig. 5 B), demonstrating that committed ILC2s are able to produce type 2 cytokine in the absence of Bcl11b, in contrast to its essential role in the early development of ILC2s. However, deletion of Bcl11b led to substantially more ILC2s that expressed KLRG1 (Fig. 5 C), which is consistent with the in vivo data (Fig. 3 B, Fig. 4 D, Fig. S3 A, and Fig. S4 A). Moreover, Bcl11b deficiency led to higher percentages of ILC2s producing IL-5 (Fig. 5 B). Therefore, Bcl11b is essential for the differentiation of ILC2s from hematopoietic progenitors but may also suppress genes that are highly expressed in mature ILC2s. Indeed, quantitative RT-PCR (qRT-PCR) confirmed up-regulation of Il5, Il13, Klrg1, Gata3, and Rora, which are highly expressed in mature ILC2s (Hoyler et al., 2012) in Bcl11b^fl/fl ILC2 cultures (Fig. 5 D).

Recent studies suggest a critical role for ILC2s in mediating protease-induced airway inflammation (for example, Halim et al., 2012a). We wished to confirm the essential role of Bcl11b in ILC2 development upon immune challenges. We purified LSKs from Bcl11b^flox/floxRosa26^{CreERT2/CreERT2}Rag1^−/− mice that were treated with Tam (Bcl11b^fl/flRag1^−/−) and transplanted them to sublethally irradiated alymphoid Rag2^−/−Il2rg^−/− recipients (CD45.1⁺). 8–10 wk after engraftment, the recipients were intranasally challenged with papain, which induces rapid IL-5 production and eosinophil infiltration in the airway (Halim et al., 2012a). Consistent with our above results (Fig. 4 J and Fig. S5 B), no ILC2s were elicited in the lung after papain challenge in Bcl11b^fl/flRag1^−/− chimeras at 84 h (Fig. 6, A and B). Papain induced eosinophil infiltration in the bronchoalveolar lavage (BAL) fluid and in the lung in control recipients, but not in the Bcl11b^fl/flRag1^−/− ones (Fig. 6, C and D). Furthermore, the papain challenge also failed to induce IL-5 or IL-13 production in the BAL fluid in the recipients from Bcl11b^fl/flRag1^−/− LSKs (Fig. 6 E).

Influenza virus infection induces type 2 immune responses and production of prototypical cytokines such as IL-5 by ILC2s (Gorski et al., 2013), which is linked to asthma exacerbation. We thus examined immune responses of BM chimeras reconstituted with LSKs upon influenza virus infection. Influenza virus infection induced expansion of ILC2s in the lung of the control recipient chimeras at day 5 after infection (Fig. 6 F and Fig. S6). In contrast, chimeras of Bcl11b^fl/flRag1^−/− LSKs completely lacked ILC2s in the lung (Fig. 6 F and Fig. S6). The control recipients, but not Bcl11b^fl/flRag1^−/− LSK chimeras, produced abundant IL-5 in the BAL fluid after influenza virus infection (Fig. 6 F). In summary, both immune challenges confirm the complete lack of ILC2 development potential in the Bcl11b-deficient hematopoietic progenitors.

We report here that Bcl11b is specifically expressed in ILCPs that are committed to ILC2s and is essential for ILC2 development. Several transcription factors have been shown to have important functions in ILC2 development. Gata3 is critical for controlling the cell fate of ILC2s (Hoyler et al., 2012; Mjösberg et al., 2012; Klein Wolterink et al., 2013) and also for all IL-7Rα–expressing ILC development (Yagi et al., 2014). Rora appears to be specifically required for inducing ILC2s (Halim et al., 2012b; Wong et al., 2012), but not RORγt-expressing ILCs (Halim et al., 2012b). However, Rora is also expressed in other ILC subsets (see microarray data in Hoyler et al., 2012). Nfil3 is shown to control type 2 Th cytokine expression (Kashiwada et al., 2011), but recent results demonstrate that it is a key regulator of the development of other ILC subsets essential for immune protection in the lung and gut (Geiger et al., 2014; Seillet et al., 2014). Bcl11b is different from these factors. It is the first transcription factor that is found specifically expressed in ILC2s and is required for development of ILC2s from hematopoietic progenitors, a function similar to its role in T cell development. Interestingly, deletion of Bcl11b in ILC2s led to higher expression of many ILC2 genes, including several transcription factors mentioned above (Fig. 5 D). This is also reminiscent of Bcl11b’s role in T cells, where deleting Bcl11b results in up-regulated expression of genes of mature T cells (Kastner et al., 2010). Further dissection of the molecular and cellular mechanisms of Bcl11b in ILC2s and ILC-specific deletion of Bcl11b using the Cre system should facilitate better understanding of ILCs and of Bcl11b’s potential role in human disease.

The Bcl11b-Tdtomato reporter mice (Bcl11b^Td/Td) and Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} mice were generated on the C57BL/6 genetic background by backcrossing to C57BL/6 mice for 11 generations. The Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} mice were crossed to Rag1^−/− mice (C57BL/6) to generate Bcl11b^flox/floxRosa26^{CreERT2/CreERT2}Rag1^−/− mice. Bcl11b germline heterozygous mutant (+/−) and the wild-type control mice (+/+) were on the 129S5 background. C57BL/6 CD45.1⁺ wild-type or Rag2^−/−Il2rg^−/− mice (C57BL/6 CD45.1; Serafini et al., 2014) were used as recipients for transplantation of BM cells or LSKs. All mice used were from colonies maintained at the research support facility of the Sanger Institute. Housing and breeding of mice and experimental procedures were done according to the UK 1986 Animals (Scientific Procedures) Act and the Animal Welfare and Ethical Review Body of the Wellcome Trust Sanger Institute.

Fluorochrome- or biotin-labeled monoclonal antibodies (clones denoted in parentheses) against B220 (RA3-6B2), CD19 (6D5), CD3ε (145-2C11), CD8α (53–6.7), TCRβ (B20.6), CD49b (DX5), TCRγδ (GL3), NK1.1 (PK136), Nkp46 (29A1.4), CD11b (M1/70), CD11c (N418), Gr1 (RB6-8C5), Ter119 (TER-119), c-kit (2B8), Flt3 (A2F10), Sca1 (D7), IL-7Rα (SB/199), CD25 (PC61), CD45.1 (A20), CD45 (30-F11), Siglec-F (E50-2440), CD27 (LG.3A10), CD244 (2B4), α4β7 (DATK32), IL-33R (RWST2-2), CCR6 (29-2L 17), IL-5 (TRFK5), IL-13 (eBio13a), IFN-γ (XMG1.2), Gata3 (L50-823), and RORγt (Q31-378) were purchased from BD, BioLegend, or eBioscience. LEGENDplex Mouse Th2 Panel (6-plex) (BioLegend) was used for BAL cytokine detection.

RBCs were removed using ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza). Cells were suspended in a solution of 2% (vol/vol) FBS in PBS. Fc receptors were blocked with anti-CD16 (2.4G2) before antibody labeling. Cells were stained with antibodies on ice for 20 min before washing. Intracellular staining was performed according to the instructions of the FOXP3 Fix/Perm Buffer Set (BioLegend). Cells were analyzed on a Fortessa (BD) or sorted on a MoFlo XDP (Beckman Coulter) according to the manufacturers’ standard operating procedures. Data were analyzed with FlowJo software, version X.0.7 (Tree Star).

BM cells were isolated by gently crushing femurs and tibias before filtration (70-µm filter). Cells from lung and siLP were prepared according to the instructions of the Lung Dissociation kit (Miltenyi Biotec) and Lamina Propria Dissociation kit (Miltenyi Biotec), respectively. The tissues were digested in a shaking water bath at 37°C for 30 min. After dissociation, leukocytes were enriched by percoll gradient centrifugation.

ILC2s were stimulated ex vivo with PMA and Ionomycin (Cell Stimulation Cocktail; eBioscience) for 4 h for IL-5 production, in the presence of the protein transport inhibitor cocktail (eBioscience). The cells were subsequently stained intracellularly for IL-5.

Mice were given intraperitoneal injections of 500 ng IL-25 or IL-33 (BioLegend) on days 0–3. On day 4, tissues were collected for analysis.

For adoptive transfer experiments, cell populations highly purified by flow cytometry were injected intravenously into sublethally irradiated (1 × 450 rad) Rag2^−/−Il2rg^−/− recipient mice (CD45.1⁺) via the tail vein. The drinking water was supplemented with antibiotics for 2 wk after irradiation.

Single-cell suspensions of BM cells from the Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} and control Bcl11b^+/+Rosa26^{CreERT2/CreERT2} mice were injected intravenously into lethally irradiated (2 × 500 rad) C57B6 recipient mice (CD45.1⁺) for reconstitution for 6–8 wk. The drinking water was supplemented with antibiotics for 2 wk after irradiation.

For hematopoietic progenitor reconstitution in vivo, the LSKs were purified from the BM of Bcl11b^fl/fl, Bcl11b^fl/flRag1^−/−, or the Bcl11b^+/+ or Bcl11b^+/+Rag1^−/− control mice. The sorted LSKs (10,000 cells) were injected into sublethally irradiated (1 × 450 rad) Rag2^−/−Il2rg^−/− recipient mice (CD45.1⁺) or with helper CD45.1⁺ BM cells (2 × 10⁵ cells) into lethally irradiated (2 × 500 rad) Rag2^−/−Il2rg^−/− recipient mice (CD45.1⁺) via the tail vein.

The mice were anesthetized with 3% isoflurane and then were intranasally administered with papain (10 µg in 40 µl PBS) every 24 h on days 0–2. 12 h after the last challenge, lungs and BAL fluid were collected and analyzed.

The BM chimeras were anesthetized with 3% isoflurane and were inoculated intranasally with influenza A virus X31 (H3N2) in 50 µl PBS of 10⁴ PFU. The virus was grown and collected from embryonated chicken eggs (48–72 h). The control (mock infected) mice were treated intranasally with allantoic fluid diluted 1:500 in PBS. On day 5 after infection, lung and BAL fluid (lavaged with 0.4 ml PBS) were collected.

The ILC2 differentiation potential of hematopoietic progenitors was performed as previously reported (Wong et al., 2012). In brief, freshly sorted CLPs from the Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} and control Bcl11b^+/+ Rosa26^{CreERT2/CreERT2} mice that were treated with Tam 4 d earlier (4.0 mg Tam by intraperitoneal injection over three consecutive days) were cultured on OP9-DL1 monolayers in the presence of 10 ng/ml IL-7 (PeproTech) and 10 ng/ml IL-33 (PeproTech) for 22 d.

For the short-term fate assay, purified Bcl11b⁻ChILPs and Bcl11b⁺ChILPs were cultured on OP9 monolayers in the presence of 25 ng/ml IL-7 (PeproTech) and 25 ng/ml Stem Cell Factor (SCF; PeproTech) for 6 d as previously described (Constantinides et al., 2014).

For deleting Bcl11b in ILC2s, sorted ILC2Ps from the BM of Bcl11b^floxfloxRosa26^{CreERT2/CreERT2} and control mice were cultured on OP9-DL1 monolayers in the presence of 20 ng/ml IL-7, 20 ng/ml SCF, and 10 ng/ml IL-2 or 20 ng/ml IL-7 plus 20 ng/ml IL-25 or plus 20 ng/ml IL-33. After 3–5 d, Tam was added in the medium to induce Bcl11b deletion in vitro. Cells were collected and analyzed 10–14 d after Tam treatment.

RNA was extracted from in vitro cultured ILC2 and reverse transcribed. TaqMan primer and probe sets (Applied Biosystems) were used for quantification of the expression of Bcl11b, Rora, Tcf7, Gata3, Il-5, Il-13, and Klrg1. Expression was quantified relative to that of Gapdh.

Data were statistically analyzed and figures were prepared using Microsoft Excel. A two-tailed Student’s t test was used throughout this work to evaluate statistical significance. Significance is indicated as follows: *, P < 0.05; **, P < 0.01.

Fig. S1 contains gating strategies of ILCs. Fig. S2 shows the gating strategies of ILCPs. Fig. S3 presents the gating strategies of ILC2s in the BM and lung of wild-type and Bcl11b heterozygous mutant mice. Fig. S4 contains gating strategies of ILCs in the Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} mice, control mice, or in BM chimeras. Fig. S5 shows gating strategies of donor LSK-derived ILCs in chimeras. Fig. S6 presents gating strategies of ILC2s in the lung of recipients engrafted with the control or Bcl11b^fl/flRag1^−/− LSKs after influenza virus (H3N2) infection.

We thank Paul Kellam’s group at the Sanger Institute for producing virus X31 (H3N2). We would also like to thank the Sanger Institute research support facility (Andrea Kirton, Georgina Owen, Paul Green, Claire Rogerson, Matthew Brealey, George Notley, and Scott Kemp) and the flow cytometry core facility (Bee-Ling Ng and Jennifer Graham) for technical assistance.

This work is supported by a grant from the Wellcome Trust (098051) to P. Liu and by grants from the National Natural Science Foundation of China (grants 31370904 and 30972691), Shanghai Science and Technology Development Fund (grant 10QA1405900), Training Program of the Excellent Young Talents of the Shanghai Municipal Health System (grant XYQ20110115), and Shanghai Municipal Science and Technology Foundation (grant 11JC1410802) to L. Lu.

The authors declare no competing financial interests.