T-Bet Controls Cellularity of Intestinal Group 3 Innate Lymphoid Cells

Innate lymphoid cells (ILC) play a significant immunological role at mucosal surfaces such as the intestine. T-bet-expressing group 1 innate lymphoid cells (ILC1) are believed to play a substantial role in inflammatory bowel disease (IBD). However, a role of T-bet-negative ILC3 in driving colitis has also been suggested in mouse models questioning T-bet as a critical factor for IBD. We report here that T-bet deficient mice had a greater cellularity of NKp46-negative ILC3 correlating with enhanced expression of RORγt and IL-7R, but independent of signaling through STAT1 or STAT4. We observed enhanced neutrophilia in the colonic lamina propria (cLP) of these animals, however, we did not detect a greater risk of T-bet-deficient mice to develop spontaneous colitis. Furthermore, by utilizing an in vivo fate-mapping approach, we identified a population of T-bet-positive precursors in NKp46-negative ILC3s. These data suggest that T-bet controls ILC3 cellularity, but does do not drive a pathogenic role of ILC3 in mice with a conventional specific pathogen-free microbiota.

INTRODUCTION CD127 + innate lymphoid cells (ILC) have been categorized into subsets based on expression of characteristic transcription factors (1). ILC1 express T-bet (encoded by Tbx21). ILC2 express GATA3 while ILC3 have a characteristic expression of RORgt. The ILC3 group has three members depending on the expression of NKp46 and CCR6. NKp46 + ILC3 express T-bet, and CCR6 -NKp46 -ILC3 have been suggested to be the precursors of these cells (2). In contrast CCR6 + ILC3 do not express T-bet and are generated following a developmental pathway that is very distinct to the other ILC (3).
There is evidence associating IBD and colitis with T-bet-expressing cells alike ILC1. We have recently reported that enhanced functionality of T-bet variants among individuals is associated with Crohn's disease and ulcerative colitis (4). Furthermore, IFNg appears to be the most critical factor driving colitis while IL-17A and IL-13 play a less important role (5)(6)(7)(8). Interestingly, these observations are corroborated by the observation that ILC1 are particularly abundant among the total amount of ILC in inflamed intestinal lamina propria of Crohn's disease patients (9)(10)(11). Furthermore, the generation of ILC1 from an NKp46 + ILC3 source has been linked to colitis development (12). We have recently observed that NKp46-dependent deletion of Tbx21 in mice leads to milder dextran sodium sulphate (DSS)-induced colitis, and this may be caused by the depletion of T-bet expressing ILC1 and ILC3 (13).
However, in contrast to these data supporting a critical pathogenic role of T-bet + ILC in colitis, we have also reported previously that germline depletion of Tbx21 in Rag-deficient BALB/c-background mice can trigger spontaneous colitis in the presence of and depending on Helicobacter thyphlonius as part of the microbiota. In these mice an elevated frequency of colitogenic IL-17A + CD90 + CD127 + ILC within the lamina propria appeared to be the driving force of the pathological phenotype. A pathogenic role of ILC3 in colitis has also been suggested by others in mouse models with H. hepaticus and anti-CD40 antibody driven colitis and even IBD patients (7,14,15). Furthermore, CCR6 + ILC3 have been linked to enhanced airway hyperreactivity in an obesity model (16). In contrast to these pathogenic roles of ILC3, several studies highlight their protective functionality in the intestine [ (17)(18)(19)(20)(21)(22)(23)(24)(25)(26) reviewed by (27)]. As such ILC3 were reported to be a critical source of protective IL-22 early during infection with Citrobacter rodentium, and depletion of CD4 + CCR6 + ILC3 using an anti-CD4 antibody led to diminished IL-22 production and enhanced bacterial tissue infiltration (28,29).
Hence, in immunocompetent patients, T-bet has the potential to be a potent drug target to control Crohn's disease and ulcerative colitis, but it needs to be established whether this approach leads to an enhanced pathogenic role of ILC3. Encouragingly, Tbx21 deficient mice on a BALB/c background do not develop spontaneous colitis (30). We have also reported previously that T-bet germline depletion in C57BL/6background mice leads to an enhanced cellularity and functionality of ILC2 which may have a beneficial role to protect from colitis with a T-bet targeting treatment (13).
In this study, we report that Tbx21 deficiency leads to greater cellularity of intestinal NKp46-negative ILC3 in Rag-sufficient and deficient mice. Interestingly, Tbx21 deficiency in these cells caused enhanced expression of IL-7R and RORgt which may play a mechanistic role in driving a greater ILC3 cellularity in immunocompetent mice. In contrast, signaling events via STAT1 and STAT4 were not critically important to drive the enhanced cellularity of T-bet-negative ILC3. Despite the greater ILC3 cellularity, Tbx21-deficient mice did not develop spontaneous colitis. We believe this work highlights the critical role of T-bet to restrain ILC3 and neutrophil cellularity in the colonic lamina propria and provides critical insight into the feasibility of drug-based targeting of T-bet as therapeutic strategy for colitis.

Generation of T-bet cre/+ Mouse
To allow the expression of the Cre-recombinase under the expression of the T-bet endogenous promoter, a T-bet knockin mouse was generated (GenOway, France). For this purpose, an IRES-Cre cassette was introduced downstream of the Stop codon of the T-bet gene, in the 3'UTR ( Figure 2A). The genomic region of interest containing the murine Tbx21 locus was isolated by PCR from 129Sv genetic background. PCR fragments were subcloned into the pCR4-TOPO vector (Invitrogen). The genomic clones (containing intron 1 to exon 6) were used to construct the targeting vector. Briefly, a 5.6-kb fragment comprising Tbx21 exon 2 and 6 and a 1.6-kb fragment located downstream of the Tbx21 exon 6 STOP codon were used to flank an IRES-Cre cassette (FRT site-PGK promoter-Neo cDNA-FRT site).

Screening of T-Bet-Targeted Embryonic Stem Cell Clones
The FseI-linearized targeting vector was transfected into C57BL/ 6 ES cells. Positive selection was started 48 h after electroporation, by addition of 200 mg/ml G418 (150 mg/ml active component; Life Technologies). Then, 275 resistant clones were isolated, amplified, and screened by PCR and further confirmed by Southern blot.

Generation of Chimeric Mice and Breeding Scheme
One floxed mutated Tbx21 ES cell clone was microinjected into albino C57BL/6 strain (C57BL/6J-Tyrc-2J/J) blastocysts, and gave rise to male chimeras with a significant ES cell contribution (as determined by the percentage of light and dark patches on their coat). After mating with C57BL/6 CMV-Flp-expressing female mice to remove the FRT-flanked Neo cassette, offspring were genotyped by PCR and Southern blot to ensure removal of the Neo cassette. PCR and Southern blot screening conditions are available on request. The mosaic excised F1 mouse was mated with C57BL/6 WT mice to obtain a pure line of Cre-expressing T-bet knock-in mice: T-bet cre/+ .

Isolation of Intestinal Leukocytes
cLP and Peyer's patch-free SI LP leukocytes were isolated using a published method (33). Briefly, the epithelium was removed by incubation in HBSS lacking Mg 2+ or Ca 2+ (Invitrogen) supplemented with EDTA and HEPES. The tissue was further digested in HBSS lacking Mg 2+ or Ca 2+ supplemented with 2% foetal calf serum (FCS Gold, PAA Laboratories), 0.5 mg/ml collagenase D, 10 mg/ml DNase I, and 1.5 mg/ml dispase II (all Roche). The LP lymphocyte-enriched population was harvested from a 40-80% Percoll (GE Healthcare) gradient interface. For neutrophil analyses leukocytes were not purified by Percoll gradient centrifugation.

Flow Cytometry
Flow cytometry was performed using a standard protocol. For ILC analyses a lineage cocktail of antibodies specific for CD3, CD45R, CD19, CD11b, TER-119, Gr-1, CD5, and FcϵRI was used. For a complete list of the antibodies used see Table 1. LIVE/DEAD TM stain (ThermoFisher Scientific Inc.) was used to determine cell viability. A FoxP3 staining kit (ebioscience) was used for intracellular staining of transcription factors and cytokines. In case of cytokine analysis, cells were prestimulated with 100 ng/ml PMA and 2 µM ionomycin in the presence of 6 µM monensin for 3-4 h prior to flow cytometry analysis. Samples were acquired using an LSRFortessa ™ cell analyser (Becton Dickinson, USA) or a Cytoflex LX ™ for the data on Stat1 -/and Stat4 -/mice. All the data were analyzed using FlowJo software (Tree Star, USA). Cell counts were determined using a fixed amount of inclusion beads (Spherotec, Inc.) as a reference in the flow cytometry tubes.

In Vivo Murine Faecal Microbiota Transplant Treatment
Faecal content extracted from the caecum of TRUC mice (31,34) was reconstituted in sterile PBS 25% glycerol prior to storage at -80°C. Mice were orally gavaged with 200 ml aliquots of this faecal solution and sacrificed after 3 weeks.
Enhanced cellularity of NKp46 -CCR6 -ILC3 in Tbx21deficient mice supported a previous report indicating that these cells are the precursors of NKp46 + ILC3 (2). Hence, the lack of Tbet could result to a developmental blockade and the accumulation of NKp46 -CCR6 -ILC3. An interlinkage of NKp46 + ILC3 and NKp46 -CCR6 -ILC3 was also highlighted in another study reporting that NKp46 + ILC3 can lose NKp46 expression (36). In order to detect NKp46 -CCR6 -ILC3 with a history of T-bet expression, we generated a mouse model that  fate-mapper mouse (T-bet FM ). As expected, cLP ILC1 defined as NKp46 + NK1.1 + T-bet + were found to be T-bet fate mapper positive (T-bet FM+ ) ( Figure 2B), confirming the functionality of the model as these cells have previously been shown to express T-bet. Interestingly, T-bet FM+ NKp46 -SI LP ILC3 were detected supporting the notion that these cells have potential to express this transcription factor ( Figure 2C).
To further evaluate the ILC3 phenotype, we analyzed the cytokine profiles of CD127 + ILC in Tbx21 -/and WT mice. As expected CD127 + ILC from Tbx21 -/mice produced very low amounts of IFNg, but surprisingly there was no altered expression of IL-17A on a per cell basis in the same mice ( Figures 3A, B). However, due to the greater cellularity of CD127 + ILC in the intestine of mice lacking T-bet (13) we anticipate more ILC expressing IL-17A. Hence, we aimed to investigate whether T-bet also controls the cellularity of cLP neutrophils (Figures 3C, D). In line with the greater cellularity of NKp46 -ILC3 in Tbx21 -/mice, there was indeed a greater neutrophilia in these mice. These neutrophils had an unaltered level of CD11b expression and granularity measured as SSC-A ( Figure 3E) indicating that these neutrophils were not activated.
We aimed to determine whether these neutrophils can be activated with a pathogenic microbiota. In order to test this Tbx21 -/mice received an oral gauvage injection of fecal microbes derived from colitic Rag2 -/-xTbx21 -/-(TRUC) mice (31,34). These Tbx21 -/mice did not show weight abnormalities during the course of 3 weeks upon fecal microbial transplant (FMT) in comparison to Tbx21 -/control mice ( Figure 3F). Furthermore, the mass of colon and spleen did not show a significant difference due to FMT 3 weeks after the treatment ( Figure 3G). FMT did also not result in altered cLP neutrophilia or cLP neutrophil activation detected by CD11b expression and granularity at this time point (Figures 3H, I). Hence, Tbx21 -/mice appeared to be resilient to spontaneous colitis driven by the pathogenic microbes used.
We have shown that untreated Tbx21 -/mice have a greater cellularity and activation of ILC2 (13). Hence, IL-5-producing ILC2 may promote an immune response counteracting to a more pathogenic response driven by ILC3 in naïve Tbx21-deficient mice (13). To explore this further we analyzed cLP eosinophilia in Tbx21 -/mice, but detected no significant difference to WT mice (Supplementary Figure 2).

T-Bet Deficient NKp46 -ILC3 Have Enhanced Expression of RORgt and CD127
Strikingly, the finding of enhanced cellularity of NKp46 -ILC3 in Tbx21 -/mice correlated with enhanced RORgt expression in total NKp46 -CCR6 -ILC3, but not in CCR6 + ILC3 ( Figure 4A). In contrast, in TRnUC mice RORgt expression was only enhanced in SI LP NKp46 -CCR6 -ILC3, but not SI LP CCR6 + ILC3 or cLP ILC3 (Supplementary Figure 3, Figure 4B). Previously, we have also reported that Tbx21 deficiency causes greater expression of the a chain of IL-7R (CD127) on total CD127 + ILC in the intestine (13). In the current study we can now pinpoint that within this Tbx21-deficient population cLP NKp46 -CCR6 -ILC3, but not CCR6 + ILC3 or ILC2 express more CD127 (Figures 4C, D). This was again in contrast to TRnUC mice as cLP and SI LP ILC3 and ILC2 in these mice did not display an altered CD127 expression in comparison to Rag2 -/mice ( Figure 4E, Supplementary Figures 3B, C). Mechanistically, we have reported previously that T-bet binds to the Cd127 locus in Th1 cells polarized in vitro (31,38). Hence, it appears that T-bet is a regulator of Cd127 and Rorc (encoding RORgt) expression at the transcriptional level which may be factors limiting the cellularity of NKp46 -CCR6 -ILC3 in Ragsufficient mice.
Mice Deficient in IFNg, IL-27Ra, STAT1, or STAT4 Have Unaltered Cellularity of NKp46 -ILC3 We further aimed to identify mediators upstream of T-bet that may be involved in limiting the cellularity of NKp46 -ILC3. Surprisingly, there was no alteration in the cellularity of NKp46 -CCR6and CCR6 + cLP ILC3 and no change in RORgt and CD127 expression in ILC3 derived from STAT1 (Stat1 -/-) or STAT4 (Stat4 -/-) deficient mice ( Figure 5A, Supplementary  Figures 4C, D). This observation was confirmed by the analysis of the same parameters in those ILC3 from mice deficient of IFNg (Ifng -/-) or IL-27Ra (Il27RA -/-), both of which cause signaling events through STAT1 ( Supplementary  Figures 4A-D). Furthermore, the absence of STAT4 but not STAT1 in the germline did result in significantly altered IL-17A expression in CD127 + CD90.2 + cLP ILC ( Supplementary  Figures 4E, F).
T-bet-deficient mice have a greater cellularity of cLP ILC2 (13). In order to correlate with these data, we analyzed ILC2 abundance in Stat1 -/and Stat4 -/mice. As observed with NKp46-negative ILC3, the cellularity of cLP ILC2 did not alter in mice deficient of either STAT1 or STAT4 and CD127 expression levels in these cells did also not change ( Figure 5B Supplementary Figure 5A). Furthermore, CD127 expression was not altered in cLP ILC2 from Il27RA -/mice (Supplementary Figure 5A). Interestingly, the potency of CD127 + CD90.2 + cLP ILC to co-produce IL-13 and IL-5 was reduced in Stat1 -/but not in Stat4 -/-(Supplementary Figure  5B). Overall, STAT1 and STAT4 did not appear to play a critical role in in controlling the cellularity of the NKp46-negative ILC3 populations and ILC2 in the cLP ( Figure 5).

DISCUSSION
There is currently no effective drug to cure IBD, and there is an urgent need to identify novel strategies of treatment. We and others have identified T-bet to be central to drive the severity of colitis in human and mice. However, targeting T-bet for instance using a small molecule inhibitor as has been tested for RORgt (39,40) requires more detailed understanding of the functional role of ILC3 in the absence of T-bet. The relevance of this has been demonstrated by the protective role of T-bet in Ragdeficient mice infected with H. thyphlonius (31,34). These data indicate that IL-17A + ILC, which could be ILC3 or inflammatory ILC2 (41), can drive colitis in the absence of T-bet and adaptive immune responses, such as regulatory T cells and sIgA, sIgM, and sIgG production.
In this study, we report that T-bet deficiency results in a very significantly increased cellularity of NKp46-negative ILC3 in Rag-deficient and Rag-sufficient naïve mice. Previously, we reported an approximate 2-fold increase in cLP ILC2 in T-betdeficient mice (13). Importantly, greater cellularity of ILC3 in Tbx21 -/mice correlated with enhanced neutrophilia in Ragsufficient Tbx21 -/mice. However, the activation of these neutrophils was not altered by T-bet-deficiency which might be due to an intact epithelial barrier preventing microbes to infiltrate the tissue. Overall, T-bet deficiency caused increased NKp46 -CCR6 -ILC3 and ILC2 cellularity in Rag-sufficient mice, but not at the cost of spontaneous colitis as observed in H. thyphlonius-infected Rag2 -/-xTbx21 -/mice (31,34).
A study by Klose et al. (2) suggested that NKp46 -CCR6 -ILC3 may be precursor cells of NKp46 + ILC3 in the intestine. Considering these data, it may be possible that the accumulation of NKp46 -CCR6 -ILC3 in T-bet-deficient mice is a consequence of the inhibited further differentiation.
Mechanistically, greater cellularity of NKp46 -CCR6 -ILC3 was correlated to greater expression of IL-7Ra and RORgt in Rag-sufficient mice. The same effect was not observed in Ragdeficient mice suggesting that in these mice those factors are unlikely driving factors of NKp46 -ILC3 cellularity in the absence of T-bet. In contrast to NKp46 -CCR6 -ILC3, an enhanced CD127 expression was not observed in CCR6 + ILC3 and ILC2 in Tbx21-deficient mice pointing to an intrinsic T-bet-dependent CD127 regulation pathway in NKp46 -CCR6 -ILC3. Interestingly, we observed that T-bet can bind to the Cd127 gene locus in CD4 T cells which may indicate that T-bet functions as a repressor of CD127 expression (31,38). In CD4 T cells, T-bet is known to inhibit transcription of Rorc (encoding RORgt), and this may be also a critical intrinsic mechanism for enhanced RORgt expression in Tbx21-deficient NKp46 -CCR6 -ILC3 (42). Interestingly, T-bet-dependent regulation of RORgt and CD127 in NKp46 -CCR6 -ILC3 was only observed in Ragsufficient C57BL/6 and not Rag-deficient BALB/c mice. Despite of this, T-bet in these Rag-deficient mice regulated cellularity of NKp46 -CCR6 -ILC3 and CCR6 + ILC3. These data indicate that T-bet may regulate the cellularity of NKp46 -CCR6 -ILC3 via further mechanisms not controlled by CD127 and RORgt expression in ILC.
We also aimed to reveal potential further signaling pathways that enhance T-bet-driven effects in NKp46-negative ILC3 and ILC2. Both IFNg and IL-27 are known to promote T-bet expression and at least pulmonary ILC3 have been reported to express the receptors for both cytokines (43)(44)(45)(46), however, we report here that the deficiency of neither of these mediators and STAT1 resulted in the enhanced cellularity of NKp46-negative ILC3 or ILC2. IFNg signals through a STAT1 homodimer, while a STAT1:STAT3 heterodimer is employed downstream of the IL-27 receptor (45,47). Furthermore, STAT4 as an important signal transmitter downstream of the IL-12 receptor regulating T-bet expression (48) did not appear to have a crucial role in the cellularity of NKp46-negative ILC3 and ILC2 as well. In addition, neither STAT1 nor STAT4 signaling events appeared to control CD127 and RORgt expression in cLP NKp46-negative ILC3. Overall, this study suggests that RORgt and IL-7R are plausible targets of T-bet to limit the cellularity of NKp46 -CCR6 -ILC3 and neutrophils in the colonic lamina propria of immunocompetent mice. This is of critical importance to evaluate potential side effects of T-bet targeted treatment of IBD.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by UK Animals (Scientific Procedures) Act 1986. Written informed consent was obtained from the owners for the participation of their animals in this study.

ACKNOWLEDGMENTS
We thank the members of the LORD laboratory for valuable discussions and critically commenting on the manuscript. In addition, we thank the BRC FlowCore at King's College and Dr Andrea Iseppon (Crick Institute, London) for technical help and Anne O'Garra (The Francis Crick Institute, London) for contributing Ifng -/mice.