# T-bet-dependent NKp46(+) innate lymphoid cells regulate the onset of T(H)17-induced neuroinflammation

Brandon Kwong, Rejane Rua, Yuanyuan Gao, John Flickinger, Yan Wang, Michael J. Kruhlak, Jinfang Zhu, Eric Vivier, Dorian B. McGavern, Vanja Lazarevic

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Brandon Kwong, Rejane Rua, Yuanyuan Gao, John Flickinger, Yan Wang, et al.. T-bet-dependent NKp46(+) innate lymphoid cells regulate the onset of T(H)17-induced neuroinflammation. Nature Immunology, 2017, 18 (10), pp.1117+. 10.1038/ni.3816 . hal-01764672

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# T-bet-dependent NKp46⁺ innate lymphoid cells regulate the onset of T<sub>H</sub>17-induced neuroinflammation

Brandon Kwong¹,⁶, Rejane Rua²,⁶, Yuanyuan Gao¹, John Flickinger Jr.¹, Yan Wang¹, Michael J. Kruhlak¹, Jinfang Zhu³, Eric Vivier⁴,⁵, Dorian B. McGavern², and Vanja Lazarevic¹

¹Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

²Viral Immunology and Intravital Imaging Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA

³Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

⁴Aix Marseille Univ, CNRS, INSERM, CIML, Marseille, France

⁵AP-HM Hôpital de la Timone, Serce d'Immunologie, Marseille, France

## Abstract

The transcription factor T-bet has been linked to increased susceptibility to systemic and organ-specific autoimmunity, but the mechanism by which T-bet expression promotes neuroinflammation remains unknown. In this study, we demonstrate a cardinal role for T-bet-dependent NKp46⁺ innate lymphoid cells (ILCs) in the initiation of CD4⁺ T<sub>H</sub>17-mediated neuroinflammation. Loss of T-bet specifically in NKp46⁺ ILCs profoundly impaired the ability of myelin-reactive T<sub>H</sub>17 cells to invade the central nervous system (CNS) tissue and protected the mice from autoimmunity. T-bet-dependent NKp46⁺ ILCs were localized in the meninges and acted as chief coordinators of meningeal inflammation by inducing the expression of pro-inflammatory cytokines, chemokines and matrix metalloproteinases, which in a concerted fashion facilitated T cell entry into CNS parenchyma. Our findings uncover a detrimental role of T-bet-dependent NKp46⁺ ILCs in the development of CNS autoimmune disease.

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Correspondence should be addressed to V.L. (lazarevicv@mail.nih.gov).

⁶Equal contribution

## AUTHOR CONTRIBUTIONS

B.K. conducted the experiments, analyzed the data and contributed to the first draft of the manuscript preparation. R.R., Y.Y.G., J.F., Y.W. conducted experiments and analyzed the data. D.B.M. contributed to the experimental design and manuscript preparation. M.J.K. performed quantitation of apoptotic cells in the CNS in a blinded manner. E.V. and J.Z. generated NKp46-Cre⁺ and T-bet ZsGreen reporter mice, respectively. V.L. designed and directed the study, conducted the experiments, analyzed the data and wrote the manuscript.

## COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.
# INTRODUCTION

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system and a leading cause of neurological disability in young adults¹. Considerable progress has been made in our understanding of immunological events that lead to the development of MS using the experimental autoimmune encephalomyelitis (EAE) model of inflammatory demyelination. Genetic targeting of interleukin 12 (IL-12) and IL-23 cytokines, necessary for the development and terminal differentiation of interferon-γ (IFN-γ)-producing CD4+ T helper 1 (TH1) and IL-17-producing T helper 17 (TH17) cells, respectively, revealed that TH17 cells, and not TH1 cells, are essential for the development of EAE²⁻⁵. Following peripheral activation, pathogenic TH17 cells migrate to the CNS and accumulate in the perivascular spaces and meninges. Here, autoantigen-driven T cell reactivation by CNS-resident antigen-presenting cells (APCs) is a prerequisite for the initiation of the inflammatory cascade by TH17 cells⁶. However, the underlying immunological factors that promote the migration of immune cells from the site of reactivation into CNS parenchyma are still not well understood.

The T-box transcription factor T-bet is critical for the development of immunopathology during EAE⁷, ⁸. T-bet is encoded by *Tbx21* and is expressed in multiple immune cell lineages of the immune system⁹. As a critical regulator of the type 1 inflammatory response, T-bet is required for the activation of host immune-defense mechanisms against infectious microorganisms¹⁰. However, excessive T-bet-regulated immune responses have been linked to the pathogenesis of immune-mediated disorders¹⁰. Although initially defined as the master regulator of the TH1 differentiation program, accumulating data indicate that T-bet is essential for the pathogenicity of TH17 cells in EAE¹¹⁻¹³. Its expression in TH17 cells is induced in response to IL-12 or IL-23 signaling¹¹, ¹², ¹⁴. T-bet promotes the functional plasticity and amplifies the inflammatory potential of TH17 cells by upregulating endogenous TGF-β3 production¹¹, ¹². Whether T-bet expression in immune cells other than TH17 cells contributes to the pathogenesis of EAE is presently unknown.

Here, we demonstrate that T-bet expression in myelin-reactive TH17 cells was necessary but not sufficient for the development of EAE. We establish a highly selective requirement for T-bet-dependent NKp46⁺ ILCs in the initiation of TH17-mediated neuroinflammation. Specifically, we found that T-bet-dependent NKp46⁺ ILCs controlled the CNS parenchymal infiltration of myelin-reactive TH17 cells by generating pro-inflammatory cytokine environment in the meninges that was necessary for the reactivation and maintenance of IL-17A-producing CD4⁺ T cells in the CNS. Our findings demonstrate a pathogenic role of NKp46⁺ ILCs in neuroinflammation and identify NKp46⁺ ILCs as a potential target for the treatment of inflammatory CNS disorders.

# RESULTS

## T-bet expression in T cells is insufficient to cause EAE

To better understand the role of T-bet in the pathogenesis of autoimmune diseases, we first wanted to determine whether T-bet expression in cells of hematopoietic origin is required for the development of organ-specific immunopathology. To address this question, we generated
conditional T-bet-deficient mice in which *Tbx21* was deleted in hematopoietic cells (*Tbx21*<sup>f/f</sup> Vav1-Cre<sup>+</sup> mice). We tested the effects of hematopoietic system-specific deletion of T-bet on susceptibility to EAE by immunizing *Tbx21*<sup>f/f</sup> Vav1-Cre<sup>+</sup> mice and their *Tbx21*<sup>f/f</sup> controls with a CNS-derived antigen, myelin oligodendrocyte glycoprotein peptide amino acids 35–55 (MOG<sub>35-55</sub>). Hematopoietic ablation of *Tbx21* gene expression resulted in significantly attenuated EAE, phenocopying germline T-bet deficiency (*Tbx21*<sup>-/-</sup>), suggesting that the pathogenic role of T-bet mapped to the cells of the hematopoietic system (Fig. 1a,b).

We therefore sought to determine in which immune cell lineage(s) T-bet expression is required to drive neuroinflammation. To this end, MOG<sub>35-55</sub> peptide-reactive T-bet-deficient (2D2 *Tbx21*<sup>-/-</sup>) and T-bet-sufficient (2D2 wild-type) CD<sup>4+</sup> T cells were activated under T<sub>H</sub>17-polarizing conditions and adoptively transferred into either T-bet-deficient (*Tbx21*<sup>-/-</sup>) or wild-type hosts. 2D2 *Tbx21*<sup>-/-</sup> T<sub>H</sub>17 cells failed to induce EAE in either *Tbx21*<sup>-/-</sup> or wild-type recipients (Fig. 1c), confirming the requirement for T-cell-intrinsic expression of T-bet in the pathology of EAE. 2D2 wild-type T<sub>H</sub>17 cells, while fully capable of inducing disease in wild-type recipients, were not pathogenic in *Tbx21*<sup>-/-</sup> recipients, demonstrating that T-cell-intrinsic expression of T-bet cells is necessary, but not sufficient, to cause EAE (Fig. 1c). Furthermore, infiltration of cells into the CNS parenchyma was severely reduced in *Tbx21*<sup>-/-</sup> recipients compared with wild-type controls (Fig. 1d), which was confirmed by histological analysis (Fig. 1e). These results revealed that T-bet expression in immune cells other than myelin-reactive CD<sup>4+</sup> T<sub>H</sub>17 cells is additionally required for the pathogenesis of autoimmune CNS disease.

## Peripheral licensing of CD4⁺ T<sub>H</sub>17 cells in *Tbx21*<sup>-/-</sup> host

Adoptively transferred myelin-reactive CD4⁺ T cells acquire the capacity to enter the CNS after effective activation within the lungs¹⁵. We asked whether peripheral licensing of adoptively transferred 2D2 wild-type T<sub>H</sub>17 cells occurred normally in the lungs of *Tbx21*<sup>-/-</sup> recipients. We found that adoptively transferred 2D2 wild-type T<sub>H</sub>17 cells egressed and proliferated normally in the spleen and lungs of both *Tbx21*<sup>-/-</sup> and wild-type recipients (Fig. 2a,b). Consistent with previous reports¹⁵, ¹⁶, myelin-reactive CD4⁺ T cells transitioned to a migratory mode in the lung by upregulating the expression of integrins α<sub>4</sub> (VLA-4 subunit) and α<sub>L</sub> (LFA-1 subunit) and the chemokine receptors CXCR4 and CCR6. Notably, the expression of these adhesion molecules and chemokine receptors on 2D2 wild-type T<sub>H</sub>17 cells was not affected by T-bet deficiency in the recipient mice (Fig. 2c). Furthermore, 2D2 CD4⁺ T cells isolated from the spleens and lungs of *Tbx21*<sup>-/-</sup> and wild-type recipients had similar percentages of cytokine-producing cells (Fig. 2d). Collectively, these data demonstrate that T-bet is not required for the licensing of 2D2 wild-type T<sub>H</sub>17 cells in the periphery.

We next sought to determine whether T-bet deficiency in recipient mice affected the effector function of 2D2 wild-type T<sub>H</sub>17 in the CNS. In addition to recovering fewer 2D2 CD4<sup>+</sup> T cells (Fig. 1c), we detected a severe reduction in the frequency of IL-17A<sup>+</sup>IFN-γ<sup>-</sup> and IL-17A<sup>+</sup>IFN-γ<sup>+</sup> 2D2 CD4<sup>+</sup> T cells in the CNS of *Tbx21*<sup>-/-</sup> recipients compared with wild-type controls (Fig. 2e). The adoptive transfer experiments with T-bet-sufficient 2D2 T<sub>H</sub>17
cells demonstrate that T-bet expression in a non-T-cell compartment controls the
parenchymal infiltration and the effector function of autoreactive CD4⁺ T<sub>H17</sub> cells in the
CNS.

T-bet-expressing cells in the CNS during EAE

We next aimed to identify T-bet-expressing cells that are necessary for the optimal activation of CD4+ T<sub>H17</sub> cells in the CNS during EAE using a previously established T-bet-ZsGreen reporter mouse<sup>17</sup>. Following EAE induction, numerous ZsGreen+ cells could be detected in the meninges, the central canal and throughout the spinal cord parenchyma (Fig. 3a). Approximately 50% of CNS-infiltrating CD45+ leukocytes expressed T-bet at the peak of disease (Fig. 3b). We identified that most T cells, NK1.1+ cells and a fraction of DCs expressed T-bet during EAE (Fig. 3c). The numbers of T-bet-expressing T cells and DCs increased in the CNS from the onset to the peak of disease. In contrast, T-bet-expressing NK1.1+ cells were detected in the CNS at the early stage of disease and their numbers remained unchanged throughout the course of disease (Fig. 3d). Therefore, we proceeded to investigate the impact of T-bet deficiency in T cells, DCs and NK1.1+ cells on the effector function of 2D2 wild-type T<sub>H17</sub> cells and their ability to induce neuropathology.

Endogenous T-bet+ T cells and DCs had no impact on EAE

Analysis of cell infiltration during EAE revealed that the majority of CNS-infiltrating CD4+
T cells were adoptively transferred 2D2 CD4+ T cells, expressing the transgenic T cell
receptor chain Vβ11, while approximately 15% of CNS-infiltrating CD4+ T cells were
endogenous (host-derived) CD4+ T cells (Supplementary Fig. 1a). To determine whether T-
bet expression in endogenous T cells affected the encephalitogenicity of adoptively
transferred 2D2 wild-type T<sub>H17</sub> cells, we transferred 2D2 wild-type T<sub>H17</sub> cells into Tbx21<sup>f/f</sup>
CD4-Cre+ recipients, which lack T-bet expression in T cells and natural killer T (NKT) cells,
and their littermate Tbx21<sup>f/f</sup> controls. Both endogenous CD4+ T cells and the transferred
2D2 (Vβ11+) wild-type T<sub>H17</sub> cells migrated efficiently into the CNS of Tbx21<sup>f/f</sup> or Tbx21<sup>f/f</sup>
CD4-Cre+ mice (Supplementary Fig. 1a). In contrast to Tbx21<sup>f/f</sup> controls, endogenous CD4+
T cells in the CNS of Tbx21<sup>f/f</sup> CD4-Cre+ contained a higher percentage of IL-17A<sup>+</sup>IFN-γ<sup>-</sup>
and a significantly lower percentage of IL-17A<sup>-</sup>IFN-γ<sup>+</sup> cells (Supplementary Fig. 1b);
nevertheless, this alteration in the cytokine profile of endogenous CD4+ T cells had no
impact on the cytokine production or pathogenicity of adoptively transferred 2D2 wild-type
T<sub>H17</sub> cells (Supplementary Fig. 1c,d).

Major histocompatibility complex class II (MHCII)-restricted antigen presentation by
meningeal and perivascular CD11c⁺ DCs and macrophages is required for the reactivation of
primed myelin-reactive T cells leading to CNS invasion and clinical disease
development⁶, ¹⁸⁻²⁰. Since 2D2 wild-type T<sub>H17</sub> cells showed suboptimal cytokine
production in the CNS of Tbx21<sup>-/-</sup> mice, we tested the importance of T-bet expression in
DCs by transferring 2D2 wild-type T<sub>H17</sub> cells into Tbx21<sup>f/f</sup> CD11c-Cre⁺ recipients, which
lack T-bet expression in DCs, and their Tbx21<sup>f/f</sup> controls. We found that DC-specific loss of
T-bet had no effect on the pathogenicity of adoptively transferred 2D2 wild-type T<sub>H17</sub> cells
in the CNS (Supplementary Fig. 2a,b). Collectively, these results show that the infiltration
and optimal effector function of adoptively transferred 2D2 wild-type T<sub>H17</sub> cells in the CNS
is not dependent on T-bet expression in endogenous (host-derived) T cells, NKT cells and DCs.

## T-bet-dependent NKp46⁺ ILCs initiate T<sub>H</sub>17-mediated EAE

To ascertain the relevance of NK1.1⁺ cells in T<sub>H</sub>17-induced neuroinflammation, we depleted NK1.1⁺ cells in *Tbx21<sup>-/-</sup>* and wild-type hosts via intravenous injections of the anti-NK1.1 depleting antibody before the adoptive transfer of 2D2 wild-type T<sub>H</sub>17 cells and repeatedly during EAE (Supplementary Fig. 3). *Tbx21<sup>-/-</sup>* recipients depleted of NK1.1⁺ cells remained resistant to EAE induction by 2D2 wild-type T<sub>H</sub>17 cells (Fig. 4a). By contrast, wild-type mice treated with anti-NK1.1 showed significantly attenuated EAE disease course compared to controls (Fig. 4b), suggesting that NK1.1⁺ cells have a pathogenic role in T<sub>H</sub>17-driven neuropathology.

T-bet is a key factor in the terminal maturation and homeostasis of NK and V<sub>α</sub>14<sup>i</sup>NKT cells and in lineage specification of ILC1 and the NKp46⁺ ILC3<sup>21-27</sup>. To evaluate the requirement for T-bet in NK cells, ILC1 and the NKp46⁺ subset of ILC3, we generated mice harboring genetic deletion of T-bet in all three ILC subsets by crossing *Tbx21<sup>f/f</sup>* mice to NKp46-Cre⁺ (also known as Ncr1-Cre+) transgenic mice. Following the adoptive transfer of 2D2 wild-type T<sub>H</sub>17 cells, mice with the NKp46-specific ablation of T-bet (*Tbx21<sup>f/f</sup> NKp46-Cre⁺*) exhibited significantly reduced mean clinical scores and percent disease incidence compared to controls (Fig. 4c,d). However, the maximum EAE disease score among symptomatic *Tbx21<sup>f/f</sup> NKp46-Cre⁺* mice was not significantly different from *Tbx21<sup>f/f</sup>* controls (Fig. 4d). These results demonstrate that the primary function of T-bet-dependent NKp46⁺ ILCs is to control the onset of T<sub>H</sub>17-mediated neuroinflammation, but once the disease is induced, they are not required for the effector stage of neuroinflammation.

## Localization of T-bet-dependent NKp46⁺ ILCs during EAE

The meninges of the CNS constitute an additional licensing checkpoint where autoantigen-driven T cell reactivation by CNS-resident APCs is a prerequisite for CNS parenchymal infiltration and subsequent demyelinating pathology⁶. Since T-bet-dependent NKp46⁺ ILCs affected the onset of T<sub>H</sub>17-mediated neuroinflammation, we hypothesized that the NKp46⁺ ILCs were strategically positioned in the meninges where they mediate the process of 2D2 wild-type T<sub>H</sub>17 cell reactivation. To assess this possibility, we have utilized a combination of high-resolution confocal microscopy and histo-cytometry to directly investigate the localization of NKp46⁺ ILCs, 2D2 CD4⁺ T<sub>H</sub>17 cells and MHCII-expressing APCs (Fig. 5a,b). The most prominent feature of the immune response in the meninges was the formation of discrete cellular clusters containing large numbers of NKp46⁺ ILCs tightly juxtaposed to both MHCII⁺ APCs and 2D2 CD4⁺ T cells. The NKp46⁺ ILC-T cell-APC clusters could be detected in the spinal cord meninges even before the onset of symptoms (score 0, Fig. 5a), indicating that they may designate the precise anatomical location of 2D2 CD4⁺ T cells reactivation. Preferential localization of NKp46⁺ ILCs within the dense clusters of MHCII⁺ APCs and 2D2 CD4⁺ T cells was additionally confirmed by histo-cytometry (Fig. 5b, blue boxes). Collectively, these observations demonstrate that NKp46⁺ ILC are localized in the meninges where they could foster 2D2 CD4⁺ T cell reactivation by influencing the function of APCs and/or autoreactive CD4⁺ T cells.
## Pathogenic function of T-bet maps to ILC1/NKp46⁺ ILC3

To investigate the significance of NKp46⁺ ILCs in the pathophysiology of CD4⁺ T<sub>H17</sub>-mediated neuroinflammation, we used multi-parameter flow cytometry to identify ILCs subsets and analyze their function in the meninges of *Tbx21<sup>f/f</sup>* and *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ recipients. In comparison to control mice, the meninges of *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice exhibited a profound deficiency in NKp46⁺ ILCs (Fig. 6a,b). We observed a significant reduction in the numbers of conventional NK cells and ILC1s while the number of NKp46⁺ ILC3s was comparable between *Tbx21<sup>f/f</sup>* and *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ recipients (Fig. 6c). T-bet-dependent NKp46⁺ ILCs predominantly produced IFN-γ and very few IFN-γ-producing NK cells and ILC1 were detected in the meninges of *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice (Fig. 6a,c). Additionally, a twofold reduction in the number of IFN-γ-producing NKp46⁺ ILC3s was detected in the meninges of *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice (Fig. 6c). Collectively, these results demonstrate that a selective loss of T-bet in NKp46⁺ ILCs resulted in meningeal depletion of NK cells and ILC1 and reduced IFN-γ-production by NKp46⁺ ILC3s.

NK cells are the numerically dominant population of T-bet-dependent NK1.1⁺ NKp46⁺ ILCs. However, the role of NK cells in EAE has been controversial. Several studies have shown that NK cells can have either pathogenic or protective functions in EAE<sup>28-32</sup>. To clarify this long-standing issue of the relevance of NK cells in EAE, we have generated NK cell-deficient *Eomes<sup>f/f</sup>* NKp46-Cre⁺ mice (Supplementary Fig. 4a,b)<sup>33</sup>. Passive transfer of 2D2 wild-type T<sub>H17</sub> cells induced EAE of equal severity in NK cell-sufficient and NK cell-deficient mice (Fig. 6d,e), demonstrating that NK cells do not have a leading role in T<sub>H17</sub>-induced neuroinflammation. Therefore, the pathogenic role of T-bet within NKp46⁺ ILCs maps to ILC1 and/or the NKp46⁺ subset of ILC3.

## NKp46⁺ ILCs regulate infiltration of CD4⁺ T<sub>H17</sub> cells

Under pathological conditions, meningeal and parenchymal infiltration of immune cells is heavily influenced by the inflammatory environment in the meninges³⁴. ILCs are a potent innate cellular source of pro-inflammatory cytokines. To test the contribution of T-bet-dependent NKp46⁺ ILCs to meningeal inflammation, we isolated the meninges from naïve mice and *Tbx21<sup>f/f</sup>* and *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice, previously injected with 2D2 wild-type T<sub>H17</sub> cells, and measured the expression of transcripts encoding pro-inflammatory cytokines by quantitative RT-PCR. Compared to naïve mice, transcripts encoding pro-inflammatory cytokines such as *p40*, *p35*, *p19*, *Il1b*, *Il6*, *Tnf*, *Lta*, *Il17a* and *Ifng* were upregulated in the meninges of *Tbx21<sup>f/f</sup>* mice (Fig. 7a). By contrast, loss of T-bet expression in NKp46⁺ ILCs resulted in significantly decreased expression of *p35*, *p19*, *Il1b*, *Il6*, *Tnf*, *Lta*, *Il17a* and *Ifng*, demonstrating that T-bet-dependent NKp46⁺ ILCs shape, directly or indirectly, inflammatory microenvironment in the meninges during T<sub>H17</sub>-mediated neuroinflammation (Fig. 7a). We hypothesized that ILC-mediated cytokine changes in the meninges, may be an important factor controlling parenchymal infiltration of immune cells. We found that meningeal accumulation of 2D2 wild-type T<sub>H17</sub> cells was not significantly different between *Tbx21<sup>f/f</sup>* and *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice. In contrast, parenchymal infiltration of 2D2 wild-type T<sub>H17</sub> cells was significantly reduced in *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice (Fig. 7b), which was confirmed by histological analysis (Fig. 7c). Integrins α₄ and α<sub>L</sub> are required for the firm adhesion and extravasation of encephalitogenic T cells while CCR6 and its ligand
CCL20 regulate the entry of T<sub>H17</sub> cells into the CNS<sup>35-37</sup>. We found that the CNS-
infiltrating 2D2 CD4<sup>+</sup> T cells expressed comparable amounts of both integrins and CCR6 in
*Tbx21<sup>f/f</sup>* and *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> mice (Fig. 7d), indicating that the lack of migration into
CNS parenchyma is not due to a T cell-intrinsic defect and that meningeal T-bet-dependent
NKp46<sup>+</sup> ILCs may regulate the parenchymal infiltration of 2D2 CD4<sup>+</sup> T cells.

T-bet-dependent NKp46⁺ ILCs could affect 2D2 CD4⁺ T<sub>H17</sub> cell parenchymal infiltration
indirectly by influencing the capacity of meningeal APCs to reactivate 2D2 CD4⁺ T<sub>H17</sub>
cells *in situ*. Activation of APCs, as measured by upregulation of MHCII, CD80 and
ICAM-1, is dependent on IFN-γ. Like *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice, *Ifng<sup>-/-</sup>* recipient mice
were protected from T<sub>H17</sub>-mediated neuroinflammation in the passive EAE model
(Supplementary Fig. 5a)³⁸. *Ifng<sup>-/-</sup>* mice showed a significant defect in the recruitment of
2D2 CD4⁺ T cells, myeloid DCs (mDCs), inflammatory monocytes (iMO) and neutrophils
to the spinal cord meninges following the adoptive transfer of 2D2 wild-type T<sub>H17</sub> cells
(Supplementary Fig. 5b,c). Additionally, resident APCs (CD206⁺ perivascular macrophages,
PVMs) failed to upregulate MHCII, CD80 and ICAM1 while mDCs showed significantly
reduced expression of CD80 and ICAM1 in the meninges of *Ifng<sup>-/-</sup>* mice (Supplementary
Fig. 5d and Supplementary Fig. 6 for gating strategy). Collectively, these data establish that
IFN-γ is of a critical importance in driving the recruitment and activation of meningeal
APCs. Next, we asked whether IFN-γ expression by NKp46⁺ ILCs was essential for optimal
activation of meningeal APCs and, consequently, for optimal reactivation of 2D2 CD4⁺ T
cells *in vivo*. In contrast to *Ifng<sup>-/-</sup>* mice, *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice showed normal
recruitment and activation of meningeal APCs following the adoptive transfer of 2D2 wild-
type T<sub>H17</sub> cells (Supplementary Fig. 7a,b), demonstrating that the expression of IFN-γ by
NKp46⁺ ILCs is not required for the recruitment or activation of meningeal APCs, and that
other cellular sources of IFN-γ could compensate for the lack of innate source of IFN-γ.

Under neuroinflammatory conditions, the gradient of chemokines produced and induced by
meningeal immune cells promotes leukocyte transmigration across the blood brain barrier
(BBB) into the CNS parenchyma³⁹. The expression of matrix metalloproteinases (MMPs)
by both resident and immune cells strongly enhances this transmigration process by
breaking down the glia limitans as well as by modulating the activity of chemokines at the
parenchymal border⁴⁰. Since the expression of MMPs and chemokines is regulated by
proinflammatory cytokines, we hypothesized that T-bet-dependent NKp46⁺ ILCs facilitated
2D2 CD4⁺ T<sub>H17</sub> cell parenchymal infiltration indirectly by inducing the expression of
MMPs and chemokines in the meninges and parenchyma, respectively. To test this
hypothesis, we examined MMP and chemokine expression in the meninges and parenchyma
of *Tbx21*<sup>f/f</sup> and *Tbx21*<sup>f/f</sup> NKp46-Cre⁺ mice following adoptive transfer of 2D2 wild-type
T<sub>H17</sub> cells by quantitative RT-PCR. Loss of T-bet expression in NKp46⁺ ILCs abrogated the
expression of major MMPs implicated in EAE such as *Mmp2*, *Mmp3*, *Mmp9*, *Mmp13*,
*Mmp14* and *Mmp19* in the meninges (Fig. 7e). Additionally, significantly fewer chemokine
mRNAs (*Ccl2*, *Ccl3*, *Ccl4*, *Ccl5*, *Ccl20*, *Cxcl9*, *Cxcl11*) were measured in the parenchyma of
*Tbx21*<sup>f/f</sup> NKp46-Cre⁺ mice compared to *Tbx21*<sup>f/f</sup> controls (Fig. 7f), indicating that optimal
chemokine gradient was not established in the parenchyma of *Tbx21*<sup>f/f</sup> NKp46-Cre⁺ mice.
Although transferred 2D2 wild-type T<sub>H17</sub> cells expressed comparable amounts of CCR6
(Fig. 7d), diminished parenchymal expression of *Ccl20* (CCR6 ligand) could account for the
migratory defect of T<sub>H17</sub> cells into the CNS parenchyma of *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> mice. These data demonstrate that T-bet-dependent NKp46<sup>+</sup> ILCs induce the expression of MMPs and chemokines which disrupt the BBB and guide the migration of 2D2 CD4<sup>+</sup> T cells out of meninges into the CNS parenchyma, respectively.

NKp46<sup>+</sup> ILCs regulate the stability of CD4<sup>+</sup> T<sub>H17</sub> cells

Functional stability and pathogenicity of CD4<sup>+</sup> T<sub>H17</sub> cells is dependent on the APC-derived
cytokines IL-1β, IL-6, IL-23, TNF-α and LT-α<sup>3, 4, 14, 41-43</sup>. Since the expression of
aforementioned cytokines was significantly reduced in the meninges of *Tbx21<sup>f/f</sup>* NKp46-
Cre<sup>+</sup> mice, we next investigated whether the maintenance of T<sub>H17</sub> responses was affected in
the CNS of *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> mice. We observed a decrease in the frequency of
IL-17A<sup>+</sup>IFN-γ<sup>-</sup> and an increase in the frequency of IL-17A<sup>-</sup>IFN-γ<sup>+</sup> 2D2 CD4<sup>+</sup> T cells both
in the meninges and parenchyma of *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> recipients compared to *Tbx21<sup>f/f</sup>*
controls (Fig. 8a,b). We additionally detected significantly less of *Il17a* transcripts in total
CNS extracts of *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> mice compared with *Tbx21<sup>f/f</sup>* controls confirming
diminished IL-17A production (Fig. 8c). These data demonstrate that the loss of T-bet
expression in NKp46<sup>+</sup> ILCs created a cytokine microenvironment in the meninges which
favored the loss of IL-17A-single producing 2D2 CD4<sup>+</sup> T cells and promoted the
differentiation of IFN-γ-single producing 2D2 CD4<sup>+</sup> T cells. Interestingly, we detected a
higher frequency of GM-CSF-producing 2D2 CD4<sup>+</sup> T cells in the meninges and parenchyma
of *Tbx21<sup>f/f</sup>* and *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> mice (Supplementary Fig. 8). Although T cell-derived
GM-CSF is required for EAE<sup>44, 45</sup>, it is not sufficient to drive neuroinflammation in *Tbx21<sup>f/f</sup>*
NKp46-Cre<sup>+</sup> mice. Importantly, the extent of myelin destruction and the frequency of
apoptotic (TUNEL<sup>+</sup>) cells was significantly decreased in the spinal cords of *Tbx21<sup>f/f</sup>*
NKp46-Cre<sup>+</sup> mice compared with *Tbx21<sup>f/f</sup>* controls (Fig. 8d,e), indicating that NKp46-
lineage specific deletion of T-bet significantly reduced the extent of immune-mediated
damage driven by autoreactive CD4<sup>+</sup> T<sub>H17</sub> cells. Collectively, these results demonstrate that
T-bet-dependent NKp46<sup>+</sup> ILCs are required for optimal reactivation, parenchymal invasion
and maintenance of myelin-reactive CD4<sup>+</sup> T<sub>H17</sub> cells in the CNS tissue.

DISCUSSION

By employing a reductionist and *in vivo* approach, we demonstrate a previously
unappreciated requirement for T-bet-dependent NKp46<sup>+</sup> ILCs in the pathogenesis of T<sub>H17</sub>-
driven neuroinflammation. EAE is a neuroinflammatory disease initiated by tissue-invading
leukocytes<sup>34</sup>. We found that myelin-reactive, pathogenic CD4<sup>+</sup> T<sub>H17</sub> cells invaded and
triggered extensive lesions throughout the CNS tissue in immunocompetent hosts while they
were absent from the CNS parenchyma of T-bet-deficient hosts. Similarly, adoptive transfer
of 2D2 wild-type T<sub>H17</sub> into mice depleted of NK1.1<sup>+</sup> cells or into mice with NKp46-lineage
specific deletion of T-bet resulted in significantly reduced disease incidence underscoring
the importance of NK1.1<sup>+</sup> NKp46<sup>+</sup> ILCs in the pathology of T<sub>H17</sub>-mediated
neuroinflammation. This group of innate lymphocytes includes NK cells, ILC1 and the
NKp46<sup>+</sup> subset of ILC3s<sup>23, 24</sup>. All three subsets of NKp46<sup>+</sup> ILCs were readily detected in
the meninges of wild-type mice during EAE, suggesting that these innate immune cells
could have a local effect on tissue-specific immune responses in the CNS.
T-bet expression impacts many aspects of NK cell homeostasis and function²², ²⁷, ⁴⁶, ⁴⁷. This raised a possibility that *Tbx21*⁻/⁻ and *Tbx21*f/f NKp46-Cre⁺ mice may be protected from T<sub>H</sub>17-mediated disease due to peripheral deficiency of NK cells. However, NK cell-deficient mice developed as severe paralysis as NK cell-sufficient mice, indicating that NK cells do not have a major effect on the immunopathogenesis of EAE and that the pathogenic function of T-bet maps to ILC1 and/or the NKp46⁺ subset of ILC3s. Our results do not exclude the possibility that NKp46⁺ ILC subsets may function redundantly. Dissecting the contributions of each NKp46⁺ ILC subset in regulating CNS-specific immune responses by generating mice with single or multiple ILC deficiencies will be an important area of future investigations.

Our findings have also raised the question of how T-bet-dependent NKp46⁺ ILCs control the onset of T<sub>H</sub>17 cell-mediated neuroinflammation. Pathology of EAE is triggered by the immune cell invasion of the CNS parenchyma, which is a highly regulated process. We think that T-bet-dependent NKp46⁺ ILCs create a microenvironment in the meninges that facilitates T cell migration across the parenchymal border, primarily through the induction of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, LT-α and IL-23), which then stimulate the expression of MMPs and chemokines. A recent study has shown that the abundance of chemokines and the expression and activity of MMPs were enhanced by encephalitogenic CD4⁺ T<sub>H</sub>17 cells, which produced high levels of IL-17A/TNFα and low levels of IFN-γ<sup>40</sup>. We observed that the microenvironment in the meninges of *Tbx21*f/f NKp46-Cre⁺ skewed the cytokine production by 2D2 CD4⁺ T cells from IL-17A to IFN-γ, indicating that the expression of chemokines and MMPs might be adversely affected by this shift in cytokine production. Consistent with that hypothesis, we found a significant reduction in the levels of chemokines and MMPs in the meninges of *Tbx21*f/f NKp46-Cre⁺ mice. Our findings also accentuate the developmental instability of CD4⁺ T<sub>H</sub>17 cells, which require constant exposure to T<sub>H</sub>17-inducing cytokines to maintain their T<sub>H</sub>17-ness.

In summary, we propose that the primary function of T-bet-dependent NKp46⁺ ILCs is to promote parenchymal infiltration of myelin-reactive CD4⁺ T cells and to maintain the functional stability of the T<sub>H</sub>17 cells by coordinating appropriate inflammatory responses in the meninges. Additionally, we demonstrate that NKp46-lineage specific deletion of T-bet was sufficient to significantly reduce the extent of immune-mediated damage driven by autoreactive CD4⁺ T<sub>H</sub>17 cells. New treatments for neuroinflammation should be designed to selectively limit responses of T-bet-dependent NKp46⁺ ILCs in the CNS.

# ONLINE METHODS

## Mice

C57BL/6 (WT), *Tbx21*⁻/⁻, *Eomes*f/f, 2D2 TCR-transgenic, Vav1-Cre⁺, CD4-Cre⁺, CD11c-Cre⁺ and R26R-YFP mice were purchased from the Jackson Laboratory. Mutant strains were previously described: NKp46-Cre⁺<sup>48</sup>, Tbet-ZsGreen reporter<sup>17</sup>, *Tbx21*f/f<sup>49</sup> and Actin-OFP<sup>50</sup>. Male and female mice were used between 7–12 weeks of age. Animal experiments were approved by the NCI Institute Animal Care and Use Committee. All mice were maintained in accordance with US National Institutes of Health guidelines.
**In vitro differentiation of 2D2 T<sub>H</sub>17 cells for adoptive transfer**

Naïve 2D2 TCR-transgenic CD4⁺ T cells from spleen and lymph nodes of 2D2 wild-type or 2D2 *Tbx21*⁻/⁻ mice were electronically sorted (CD4⁺V<sub>β</sub>11⁺CD62L<sup>hi</sup>), and activated under T<sub>H</sub>17 polarizing conditions with the following: anti-CD3 (2.5 µg/ml) (145-2C11, BioXCell), anti-IL-4 (20 µg/ml) (11B11, BioXCell), anti-IFN-γ (20 µg/ml) (XMG1.2, BioXCell), mIL-6 (30 ng/ml) (Miltenyi Biotec), hTGF-β1 (3 ng/ml) (Miltenyi Biotec) in the presence of irradiated wild-type splenocytes at 5:1 ratio. After 60 h of activation, mIL-23 (10 ng/ml; R&D Systems) was added. On day 5 of culture, cells were reactivated on plates pre-coated with 2 µg/ml of anti-CD3 and anti-CD28 (PV1, BioXCell), for an additional 48 h, prior to adoptive transfer.

**Active and passive induction of EAE and disease analysis**

Active EAE was induced by immunization with the myelin oligodendrocyte glycoprotein (MOG) peptide amino acids 35–55 (MEVGWYRSPFSRVVHLYRNGK). 100 µg MOG<sub>35-55</sub> peptide was emulsified in complete Freund's adjuvant supplemented with *M. tuberculosis* extract H37Ra (Difco), and injected subcutaneously. Mice received 150 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2. Passive transfer EAE was induced by injecting 2D2 T<sub>H</sub>17-polarized cells (as described above) intravenously into recipient mice at 5–7.5 × 10<sup>6</sup> cells/mouse. For NK depletion experiments, 0.5 mg/dose of purified anti-NK1.1 (clone PK136, BioXCell) was injected intravenously into mice on days –5/–3/–1 prior to 2D2 adoptive transfer, and additionally every 3 days from day 1 post-2D2 transfer. Classical EAE symptoms were scored daily according to standard criteria: 0, asymptomatic; 1, flaccid tail; 2, hind limb weakness and impaired righting ability; 3, hind limb paralysis; 4, front and hind limb paralysis; 5, moribund or death.

**Isolation of CNS parenchymal and meningeal cells**

For isolation of CNS-infiltrating cells for flow cytometry, brain and spinal cord tissues were minced and digested using 2.5 mg/ml collagenase D (Roche) and 1 mg/ml DNase I (Roche) for 30 min at 37 °C. Tissues were mechanically disrupted using a gentleMACS Dissociator (Miltenyi), filtered through 70 µm mesh strainers, and centrifuged through a Percoll density gradient (38% and 70%). Mononuclear cells in the interphase were removed, washed and resuspended in culture medium for analysis by flow cytometry. For isolation of spinal cord meningeal cells, mice were transcardially perfused with PBS and the meninges was extracted from the spinal column after removal of the spinal cord parenchyma. Meninges tissues were digested using 2.5 mg/ml collagenase D and 0.1 mg/ml DNase I for 30 min at 37 °C, then filtered through 70 µm mesh strainers to generate single-cell suspensions.

**Antibodies**

The following antibody clones were used for flow cytometry: B220 (RA3-6B2); CCR6 (29-2L17); CD11b (M1/70); CD11c (HL3); CD16/32 Fc Block (2.4G2); CD206 (C068C2); CD3ε (145-2C11); CD4 (RM4-5); CD45 (30-F11); CD45.2 (104); CD49b (DX5); CD54/ICAM-1 (YN1/1.7.4); CD80 (16-10A1); CXCR4 (2B11); F4/80 (BM8); IFN-γ (XMG1.2); IL-17A (TC11-18H10.1); Itgα<sub>4</sub>/CD49d (R1-2); Itgα<sub>L</sub>/CD11a (M17/4); Ki67 (B56); Ly-6C (HK1.4); Ly-6G (1A8); I-A/I-E (M5); NK1.1 (PK136); NKp46 (29A1.4); Thy1.2 (30-H12);
TCRβ (H57); Vβ11 TCR (RR3-15); RORγt (AFKJS-9); CD127/IL-7Ra (A7R34); Gr1 (RB6-8C5); TCRγδ (GL3). The following antibody clones were used for immunohistology: CD4 (RM4-5); myelin basic protein (SMI-94).

## Flow cytometry

For intracellular cytokine staining of cell suspensions from the spleen, lung, or CNS, cells were first stimulated *ex vivo* for a total of 4 h with PMA (50 ng/ml; Sigma) and ionomycin (1 µM; Sigma), including the addition of monensin (3 µM) to block secretion in the last 2 h of stimulation. Cells were stained with fixable live/dead dye according to the manufacturer's protocol (Life Technologies), then stained with antibodies against surface markers in the presence of anti-CD16/32 Fc Block (2.4G2) for 30 min at 4 °C. After surface marker staining, cells were fixed and permeabilized in Cytofix/Cytoperm solution (BD Biosciences) for 30 min prior to intracellular staining of cytokines for 30 min at 4 °C. Ki67 intranuclear staining was performed following fixation and permeabilization using the Foxp3/Transcription Factor Staining kit from eBioscience. Samples were acquired on a BD LSRFortessa or LSRII flow cytometer (BD Biosciences), and FlowJo software (TreeStar) was used for data analysis.

## Immunohistology and TUNEL quantification

Mice were transcardially perfused with 2% or 4% paraformaldehyde (PFA) for *in vivo* fixation. Spinal cords were extracted and placed in 2% PFA at 4°C for 12 h of additional fixation, then transferred to 30% sucrose for >24h before embedding in Tissue-Tek OCT compound (Sakura) for cryopreservation. Frozen tissues were cryosectioned into 12 µm slices for immunostaining. Prior to staining, sections were re-fixed with 1% PFA for 10 min, washed with PBS, and blocked by incubating with 1% bovine serum albumin and 5% normal goat serum for 2 h at 23 °C. Primary antibodies against CD4 and myelin basic protein (MBP) were incubated for at least 12 h at 4 °C. Fluorescently labeled secondary goat antibodies (Jackson Immuno) against the relevant species were incubated for 2 h at 23 °C. After washing and counterstaining with DAPI (Life Technologies), slides were mounted with Vectashield Antifade Mounting Medium (Vector Laboratories) and sealed. For negative staining controls, sections were incubated with secondary antibodies after omitting primary antibodies. For the detection of apoptotic cells by TUNEL assay, the ApopTag Fluorescein *In Situ* Apoptosis Detection Kit (Millipore) was used according to the manufacturer's protocol. Images were acquired using a Zeiss LSM880 confocal microscope and processed using Zen imaging software (Zeiss). Multiple slices from each experimental mouse were examined to ensure the reproducibility of histological staining. The quantification of TUNEL+ cells from histological images was performed using Zen imaging software in a blinded manner by the EIB Microscopy Facility (NCI), normalized to the total tissue section area, and expressed as # cells/mm².

## Histo-cytometry

z-stack images of whole mount spinal cord meninges were collected using Olympus FV1200 laser scanning confocal microscope using the 405, 488, 559, and 635 nm laser lines, and objectives with 4× magnification (0.16 NA), and 20× magnification (0.75 NA), 10 µm-step size at a voxel density of 1024×1024. Fluorophore emission was collected on 4 side window
PMT with sequential laser excitation to minimize spectral spillover. The extended field of view images were acquired using the multi-area timelapse module of the image acquisition software (Olympus fluoview Ver.4.2a). The grid of images was stitched using the same module to a single large image. DAPI, YFP, OFP and Alexa-647 channels were collected to detect nucleated cells, NKp46⁺ cells, 2D2 CD4⁺ T cells and I-A/I-E⁺ APCs, respectively. For YFP, OFP and Alexa-647 channels, threshold identification, voxel gating and spot creation without background subtraction were performed using the Spot Creation Wizard (Imaris software) that provided automatic threshold identification of the positively stained cells based on the ‘Quality’ parameter. The estimated diameter used for thresholding was set as 15 µm. For the spots corresponding to each cell type, all 4 channel statistics (DAPI, YFP, OFP, Alexa-647) were exported into Excel (Microsoft) and converted to a csv file for direct visualization in FlowJo v10 (Treestar). For each cell type, DAPI-positive spots were then selected against debris based on value-based visual thresholding and were discriminated on a biexponential-scale. Those actual cells were then plotted in color on a 10⁻²–10⁴ biexponential-scale in the dot-plots shown in Fig. 5. Irrelevant populations (NKp46⁺ spots for I-A/I-E⁺ APCs and 2D2 CD4⁺ T dot-plots, and I-A/I-E⁺ spots for NKp46⁺ dot-plot) before DAPI-positive selection are plotted in grey to show negative populations.

## Quantitative RT-PCR on tissue homogenates

Mice were transcardially perfused with PBS, and the spinal cord parenchyma and spinal cord meningeal tissue were harvested. Tissues were immediately immersed in QIAzol Lysis Reagent (Qiagen) with zirconia/silica beads (Biospec), mechanically homogenized, and stored at -80 °C. RNA isolation was performed by phenol-chloroform extraction, and reverse transcription was performed using MultiScribe Reverse Transcriptase (Thermo Fisher). RT-PCR was carried out using the SYBR Green PCR system (Thermo Fisher), and analyzed on a QuantStudio 6 Flex instrument (Thermo Fisher). mRNA expression was calculated by the ΔCt method, relative to housekeeping genes *Hprt* or *Rpl13*. RT-PCR primer sequences are listed in the Supplementary Table 1.

## Statistics

Statistical significance was calculated by unpaired two-tailed Student's *t*-test or 2-way ANOVA, as noted in figure legends, using GraphPad Prism software. All data are presented as mean ± standard error of mean (s.e.m.), unless otherwise noted.

## Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

## Acknowledgments

We thank Y. Belkaid and A. Singer for critical review of the manuscript, S. Sharrow, L. Granger and A. Adams for flow cytometry and cell sorting, T. Loo for technical assistance and the members of the A. Singer and H. Park laboratories for discussions. S. Reiner (Columbia University) generously provided *Tbx21f/f* mice. This study was supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research (ZIA BC 011431 and ZIA BC 011432) and National Institute of Neurological Disorders and Stroke.
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<figure><img src="image_2.png" /></figure>

**Figure 1.**

T-bet expression in T cells is insufficient to cause EAE. (a–b) Mean clinical scores in wild-type (WT) versus *Tbx21*−/− mice (a), or *Tbx21*<sup>f/f</sup> versus *Tbx21*<sup>f/f</sup> Vav-Cre<sup>+</sup> mice (b), immunized with MOG<sub>35-55</sub>/CFA and pertussis toxin. (c) Mean clinical scores in *Tbx21*−/− or WT mice receiving 5 × 10<sup>6</sup> 2D2 *Tbx21*−/− or 2D2 WT T<sub>H</sub>17 cells. (d) Enumeration of total CNS-infiltrating mononuclear cells or CNS-infiltrating 2D2 CD<sup>4+</sup> T cells (CD<sup>4+</sup>V<sub>β</sub>11<sup>+</sup>) at the peak of EAE disease (day 15–17 post-transfer), in *Tbx21*−/− or WT recipients of 2D2 WT T<sub>H</sub>17 cells (5–7.5 × 10<sup>6</sup>), analyzed by flow cytometry. (e) DAPI staining of spinal cord longitudinal sections from a naïve healthy mouse or *Tbx21*−/− or WT recipients of 2D2 WT T<sub>H</sub>17 cells (7.5 × 10<sup>6</sup>). Scale bar, 200 µm. *, P < 0.0001 (two-way ANOVA (a–c) or two-tailed Student's t-test (d)). Data are combined from three independent experiments (a; mean ± s.e.m. of *n* = 30 mice per group), four independent experiments (b; mean ± s.e.m. of *n* = 28 mice per group), five independent experiments (c; mean ± s.e.m. of *n* = 15–20 mice per group) and 12 independent experiments (d; mean ± s.e.m. of *n* = 54, 57 mice per group). Histological images are representative of three independent experiments (*n* = 6, 9 mice per group).
<figure><img src="image_3.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.**</figcaption></figure>

Peripheral licensing of CD4+ T<sub>H17</sub> cells in *Tbx21<sup>-/-</sup>* host. (a) Time-course analysis quantifying the number of 2D2 CD4+ T cells (CD4+V<sub>β</sub>11+) in the spleens or lungs of *Tbx21<sup>-/-</sup>* or WT mice, following adoptive transfer of 7.5 × 10<sup>6</sup> 2D2 WT T<sub>H17</sub> cells. (b) Ki67 intracellular staining of 2D2 CD4+ T cells (CD3+CD4+V<sub>β</sub>11+) trafficking through the spleens or lungs of *Tbx21<sup>-/-</sup>* or WT mice five days post-adoptive transfer (n = 5 mice per group). Histograms are overlaid with Ki67 expression in endogenous WT (BL6) CD4+ T cells (shaded). (c) Expression of migratory receptors on 2D2 CD4+ T cells trafficking through the spleens or lungs of *Tbx21<sup>-/-</sup>* or WT mice five days post-adoptive transfer. Histograms are overlaid with expression detected on endogenous WT (BL6) CD4+ T cells (shaded). (d) Intracellular cytokine staining of 2D2 CD4+ T cells from the spleens or lungs of *Tbx21<sup>-/-</sup>* or WT mice, five days post-adoptive transfer. (e) Intracellular cytokine staining of 2D2 CD4+ T cells from the CNS of *Tbx21<sup>-/-</sup>* or WT mice at the peak of EAE disease (day 15–17 post-transfer). *, *P* < 0.0001 (two-tailed Student's *t*-test). Data are representative
of two independent experiments (a,d; mean ± s.e.m. of n = 6 mice per group), three
independent experiments (c; n = 10 mice per group) or five independent experiments (e;
mean ± s.e.m. of n = 27 mice per group).
<caption>a</caption>

DAPI T-bet Zs-Green

<figure><img src="image_8.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 3.**</figcaption></figure>

T-bet-expressing cells in the CNS during EAE. (a) DAPI staining of spinal cord longitudinal sections from T-bet-ZsGreen reporter mice immunized with MOG35-55/CFA and pertussis toxin, harvested at the peak of EAE disease (day 14 post-immunization). Inset images show ZsGreen reporter+ cells in the meninges (m) and central canal (c) of the spinal cord. Scale bar, 50 µm. (b) Flow cytometric analysis of the CNS of WT (BL6) or T-bet ZsGreen reporter mice immunized with MOG35-55/CFA and pertussis toxin. Analysis is gated on total live cells. (c) Analysis of T-bet ZsGreen expression in CNS-infiltrating immune cell populations from T-bet ZsGreen reporter mice immunized with MOG35-55/CFA and pertussis toxin. Immune cell subsets were gated on live CD45+ cells, then further identified as follows: T cells, MHCII-CD3+; dendritic cells, CD3-MHCII+CD11c+; NK1.1+ innate cells, CD3-MHCII-NK1.1+; B cells, CD3-MHCII+B220+; inflammatory monocytes, CD3-CD11b+Ly6ChiLy6G-; neutrophils, CD3-CD11b+Ly6CintLy6G+. (d) Quantitation by flow cytometry of T-bet-expressing CD45+ immune cells, T cells, dendritic cells or NK1.1+ innate cells from the CNS of T-bet ZsGreen reporter mice immunized with MOG35-55/CFA and pertussis toxin. CNS-infiltrating cells were harvested at the onset (day 7) or peak of
EAE disease (day 13–14 post-immunization) and stained for lineage-specific markers as in (c). Data in (b–d) are combined from 2–3 independent experiments, using 5 pooled mice per group per experiment; data are presented as mean ± s.e.m (d). Histological images are representative of 2 independent experiments (n = 4 mice).
<figure><img src="image_12.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_15.png" /></figure>

**Figure 4.**

T-bet-dependent NKp46⁺ ILCs initiate T<sub>H</sub>17-mediated EAE. (a–b) Mean clinical scores of *Tbx21<sup>-/-</sup>* and WT mice following the adoptive transfer of 7.5 × 10<sup>6</sup> 2D2 WT T<sub>H</sub>17 cells, with anti-NK1.1-mediated depletion (αNK1.1) or PBS control injections performed before and during EAE disease. Detailed description of the anti-NK1.1 (clone PK136) depletion regimen is described in Supplementary Fig. 3. (c) Mean clinical scores of *Tbx21<sup>f/f</sup>* or *Tbx21<sup>f/f</sup>* NKp46-Cre<sup>+</sup> mice following adoptive transfer of 7.5 × 10<sup>6</sup> 2D2 WT T<sub>H</sub>17 cells. (d) The disease incidence in each independent experiment (percentage of mice demonstrating EAE symptoms) and the maximum clinical score among EAE symptomatic mice only. NS, *P* > 0.05; *, *P* < 0.01; ** *P* < 0.0001 (two-way ANOVA (b,c) or two-tailed Student's *t*-test (d)). Data are representative of 2 independent experiments (a; mean ± s.e.m. of *n* = 12 mice per group), four independent experiments (b; mean ± s.e.m. of *n* = 25, 27 mice per group) and 8 independent experiments (c,d; mean ± s.e.m. of *n* = 37, 49 mice per group).
a

Adoptive transfer:

2D2 CD4+ OFP T<sub>H</sub>17 cells → NKp46-Cre+ Rosa26-YFP recipients

<figure><img src="image_16.png" /></figure>

b

<figure><img src="image_17.png" /></figure>

**Figure 5.**

Localization of T-bet-dependent NKp46⁺ ILCs during EAE. (a) A total of 7.5 × 10⁶ 2D2 CD4⁺ OFP⁺ T<sub>H</sub>17 cells were adoptively transferred into NKp46⁺ reporter mice (NKp46-Cre⁺ *Rosa26-loxP*-STOP-<i>loxP</i>-YFP). Spinal cord meninges were isolated from NKp46⁺ reporter mice before the onset of symptoms (score 0) and during the peak of disease (score 3) and stained with DAPI and anti-MHCII (I-A/I-E). Inset images show clusters of NKp46⁺ (green) and 2D2 CD4⁺ T cells (white), without (left) and with (right) MHCII (I-A/I-E) staining. Scale bars, 500 mm for main images or 40 µm for insets. (b) Spots statistics from the whole mount shown in (a) (score 3) were exported and plotted for identification of distinct immune subsets. NKp46-YFP⁺, 2D2 CD4⁺ OFP and I-A/I-E voxel gating allowed identification and spatial visualization of the NKp46⁺ ILCs, 2D2 CD4⁺ T cells and MHCII-
expressing APCs, respectively, by histo-cytometry (as described in **Methods**). Histological
analysis is representative of 2 independent experiments (n = 3 mice for score 0 and n = 4
mice for score 3).
<figure><img src="image_18.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 6.**</figcaption></figure>

Pathogenic function of T-bet maps to ILC1/NKp46⁺ ILC3. (a) Flow cytometry analysis of meningeal immune cells following adoptive transfer of 7.5 × 10⁶ 2D2 WT T_H17 cells into Tbx21f/f or Tbx21f/f NKp46-Cre⁺ mice. Cells were stimulated with PMA and ionomycin for 3 h in the presence of monensin in the last 1.5 h of stimulation. Stimulated cells were stained with viability dye, antibodies to lineage markers (TCRβ, TCRγδ, B220, Gr1) and anti-CD45, anti-NKp46, anti-IL-7Rα, anti-CD49b (DX5), anti-RORγt, anti-IL-17A and anti-IFN-γ. Production of IL-17A and IFN-γ by meningeal NK cells (gated on Lin⁻CD45⁺NKp46⁺RORγt⁻DX5⁺IL-7Rα⁻), ILC1 (gated on
Lin⁻CD45⁺NKp46⁺RORγt⁻DX5⁻/loIL-7Rα⁺) and NKp46⁺ ILC3 (gated on Lin⁻CD45⁺NKp46⁺RORγt⁺IL-7Rα⁺) was measured by intracellular cytokine staining. (b) The box-and-whiskers plot depict the frequency of NKp46⁺ ILCs within Lin⁻CD45⁺ population in the meninges of Tbx21f/f or Tbx21f/f NKp46-Cre⁺ mice. (c) Absolute numbers of NK cells, ILC1 and NKp46⁺ ILC3 and IFN-γ-producing NK cells, ILC1 and NKp46⁺ ILC3 (gated as in a) in the meninges of Tbx21f/f or Tbx21f/f NKp46-Cre⁺ mice. (d–e) Mean clinical scores, percent disease incidence and maximum clinical score of Eomesf/f or Eomesf/f NKp46-Cre⁺mice following adoptive transfer of 7.5 × 10⁶ 2D2 WT T_H17 cells. NS, *P > 0.05; **P < 0.05; ns, *P < 0.05; **P < 0.001* (two-tailed Student's t-test (b,c,e) or two-way ANOVA (d)). Data are combined from two independent experiments (b,c; n = 9, 10 mice per group) or three independent experiments (d,e; n = 29, 35 mice per group).
<figure><img src="image_25.png" /><figcaption>Figure 7.</figcaption></figure>

NKp46⁺ ILCs regulate infiltration of CD4⁺ T<sub>H17</sub> cells. (a) Quantitative RT-PCR analysis of genes encoding pro-inflammatory cytokines in the spinal cord meninges of *Tbx21<sup>f/f</sup>* or *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice following the adoptive transfer of 7.5 × 10<sup>6</sup> 2D2 WT T<sub>H17</sub> cells. Gene expression is presented relative to the housekeeping gene *Hprt*, and normalized to transcript abundance from naïve WT (BL6) mice. (b) Absolute number of 2D2 CD4⁺ T cells (CD4⁺Vβ11⁺) in the spinal cord meninges and CNS parenchyma of *Tbx21<sup>f/f</sup>* or *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice as quantitated by flow cytometry. (c) Immunohistological analysis of longitudinal spinal cord sections from *Tbx21<sup>f/f</sup>* or *Tbx21<sup>f/f</sup>* NKp46-Cre⁺ mice following the adoptive transfer of 2D2 WT T<sub>H17</sub> cells. Tissue sections were stained with DAPI and anti-CD4. Scale bars, 200 µm for main images or 50 µm for insets. (d) Expression of integrins
α₄, α_L and CCR6 on CNS-infiltrating 2D2 CD4⁺ T cells (day 18–20 post-adoptive transfer). Bar graphs depict the average mean fluorescence intensity (MFI) of α₄ and α_L staining and mean percentage of CCR6⁺ 2D2 CD4⁺ T cells. (e–f) Quantitative RT-PCR analysis of genes encoding matrix metalloproteinases (MMPs) in the spinal cord meninges and chemokines in the spinal cord parenchyma of *Tbx21fl/fl* or *Tbx21fl/fl* NKp46-Cre⁺ mice (quantified as in a). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t-test). Data are combined from two independent experiments (a,f; mean ± s.e.m. of n = 11 mice per group, d; n = 6, 7 mice per group), three independent experiments (e; mean ± s.e.m. of n=15, 19 mice per group) or four independent experiments (b; mean ± s.e.m. of 17 mice per group). Histology images are representative of 2 independent experiments (n = 6 mice per group).
<figure><img src="image_26.png" /><figcaption>**Figure 8.**</figcaption></figure>

NKp46⁺ ILCs regulate the stability of CD4⁺ T<sub>H17</sub> cells. (a) Intracellular cytokine staining of 2D2 CD4⁺ T cells isolated from the meninges and CNS parenchyma of *Tbx21<sup>fl/fl</sup>* or *Tbx21<sup>fl/fl</sup>* NKp46-Cre⁺ mice following adoptive transfer of 7.5 × 10<sup>6</sup> 2D2 WT T<sub>H17</sub> cells. (b) Box-and-whiskers plots of cytokine producing 2D2 CD4⁺ T cells isolated from the CNS parenchyma of *Tbx21<sup>fl/fl</sup>* or *Tbx21<sup>fl/fl</sup>* NKp46-Cre⁺ mice. (c) Quantitative RT-PCR analysis of genes encoding IL-17A and IFN-γ in the CNS parenchyma of *Tbx21<sup>fl/fl</sup>* or *Tbx21<sup>fl/fl</sup>* NKp46-Cre⁺ mice (quantified as in Fig. 7a). (d–e) Longitudinal spinal cord sections from *Tbx21<sup>fl/fl</sup>* or *Tbx21<sup>fl/fl</sup>* NKp46-Cre⁺ mice were stained with anti-myelin basic protein antibody. TUNEL staining was performed to quantitate apoptotic cell death in the spinal cord tissue, and results were normalized to the total tissue area analyzed. Scale bars, 200 µm (d) or 100 µm (e). *, P < 0.05; **, P < 0.001 (two-tailed Student's t-test). Data are representative of 2 independent experiments (meninges) or five independent experiments (parenchyma) (a), or combined from five independent experiments (b; n = 24, 28 mice per group) and 2 independent experiments (c; n = 10, 11 mice per group). Histology images are representative of 2
independent experiments, while TUNEL analysis was performed on 2 sections per mouse,
with 3-4 mice per group (d-e).