Eomes Transcription Factor Is Required for the Development and Differentiation of Invariant NKT Cells

ARTICLE

https://doi.org/10.1038/s42003-019-0389-3 OPEN Eomes transcription factor is required for the development and differentiation of invariant NKT cells

Kanako Shimizu1,3, Yusuke Sato1,3, Masami Kawamura1, Hiroshi Nakazato1, Takashi Watanabe2,

1234567890():,; Osamu Ohara2 & Shin-ichiro Fujii1,2

Eomes regulates the differentiation of CD8+ T cells into effector and memory phases. However, its role in invariant (i)NKT cells remains unknown. Here, we show the impact of Eomes on iNKT cells in the thymus and peripheral tissue using conditional knockout (Eomes- cKO) mice. In the thymus, CD1d-tetramer+CD24+CD44−NK1.1−CD69+stage 0 iNKT cells express higher levels of Eomes than the other iNKT stages. We also found that Eomes regulates NKT1 cell differentiation predominantly. Interestingly, the expression of Eomes in the steady state is low, but can be upregulated after TCR stimulation. We also showed epigenetic changes in the Eomes locus after activation. In addition, vaccination of C57BL/6, but not Eomes-cKO mice with iNKT ligand-loaded dendritic cells generated KLRG1 +iNKT cells in lung, characterized as effector memory phenotype by transcriptome proﬁling. Thus, Eomes regulates not only the differentiation of NKT1 cells in the thymus, but also their differentiation into memory-like KLRG1+iNKT cells in the periphery.

1 Laboratory for Immunotherapy, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Kanagawa 230-0045, Japan. 2 Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Kanagawa 230-0045, Japan. 3These authors contributed equally: Kanako Shimizu, Yusuke Sato. Correspondence and requests for materials should be addressed to K.S. (email: [email protected]) or to S.-i.F. (email: [email protected])

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3

nvariant (i)NKT cells express an invariant T cell antigen we analyzed T cell-specific Eomes-deficient mice, specifically receptor (TCR) α-chain and recognize a complex consisting of CD4-Cre Eomesf/f (Eomes cKO) mice. Conventional CD4+ T cell I + the antigen-presenting MHC-like molecule CD1d and a and CD8 T cell development in the thymus of Eomes cKO mice glycolipid1,2. Recent discoveries of key transcription factors and was unaffected, as assessed by cell numbers and ratios of thy- gene-expression programs have contributed to a deeper under- mocyte subsets, which were similar to those of wild type mice standing of the iNKT cell lineage3. In the commitment to the (Supplementary Fig. 1a). The CD4+ to CD8+ T cell ratio in the NKT cell lineage, iNKT precursors are positively selected by CD4 spleen of Eomes cKO mice was also relatively normal (Supple- +CD8+ thymocytes which express CD1d-glycolipod complexes4. mentary Fig. 1a), consistent with a previous report15. However, After the selection, the TCR signaling initially activates the cal- the frequency of iNKT cells in thymus, spleen, and liver of Eomes cineurin pathway and stimulates the transcription factors, NFAT cKO mice was lower than in WT controls (Fig. 1a). Closer ana- and Egr25. Then, Egr2 activates PLZF as the iNKT cell master lysis of the thymus revealed that the frequency and absolute regulator and also induces the common β subunit of the IL-2/IL- number of CD24−CD44−NK1.1-stage 1 and CD44+NK1.1− 15 receptor (CD122). This leads to the subsequent steps of iNKT stage 2, iNKT cells were increased, but those of CD44+NK1.1+ cell development, composed of cytokine expression and pro- stage 3 iNKT cells were decreased (Fig. 1b, c and Supplementary liferation in response to the IL-15-CD122 axis6. Functional sub- Fig. 2a). Since thymic iNKT cells contain CD4+CD8− single sets of iNKT cells, NKT1, NKT2, and NKT17, have been reported positive and CD4−CD8− double negative subsets, we analyzed to arise during the thymus to peripheral differentiation stages. CD4 and CD8 expression. No change in the ratio of CD4 single The key transcription factors for NKT1, NKT2 and NKT17 positive and CD4−CD8− double negative subsets was observed in development have been identified as T-bet, Gata3 and Rorγt, Eomes cKO mice (Supplementary Fig. 2b). Of note, Eomes cKO respectively3. However, these iNKT cell subsets can also undergo mice expressed higher levels of Vα14TCR than WT controls, further differentiation in the periphery. clearly indicating that the development of iNKT cells in the We previously reported that a population of Klrg1+ iNKT cells thymus was impaired (Fig. 1d and Supplementary Fig. 3). These elicited after administration of α-GalCer-pulsed DCs (DC/Gal) results suggested that the terminal maturation of iNKT cells in are long-lived. These cells predominantly express the NKT1-like the thymus may depend on Eomes expression. Recent studies cytokine IFN-γ, cytotoxic molecules (FasL and granzyme A/B) have suggested that iNKT cell precursors in the thymus receive and in particular express Eomes. Moreover when transferred to the strongest TCR signal during selection rather than mature iNKT cell-deficient mice, they can respond to the same antigen iNKT cells16,17. Therefore, we assessed the level of Eomes in NKT quickly and robustly as a secondary memory type response7. stages 0 to 3 in the thymus by quantitative PCR (qPCR). To However, the molecular mechanism by which the Klrg1+ iNKT isolate stage 0 iNKT cells as iNKT precursor cells, we sorted population is generated after administration of DC/Gal remains CD1d-tetramer+CD24+CD44−NK1.1−CD69+ cells. As shown in unknown. Fig. 1e, stage 0 iNKT cells showed the highest expression of Eomesodermin (Eomes) is a T-box transcription factor with Eomes compared to the other stages. Thus, the expression of high homology to T-bet and is expressed by activated CD8+ Eomes may be related to TCR signaling in the thymus. T cells as well as in resting and activated NK cells8. Eomes is not expressed by naïve T cells, but plays an important role in the formation of conventional memory CD8+ T cells9. In particular, Loss of Eomes decreases NKT1 cells. Recently, iNKT cells have Eomes favours the development of central memory cells, char- been categorized into three lineages based on their master tran- acterized by longer survival and the important potential for scription factors. T-bet is critical for iNKT cell terminal homeostatic proliferation10. In addition, Eomes functions as a maturation and NKT1 generation, whereas Gata3 and Rorγt are master regulator of cell-mediated immunity capable of control- crucial transcription factors for NKT2 and NKT17 differentiation, ling the expression of genes encoding essential effector molecules, respectively3,17–20. We analyzed the expression of several tran- such as IFN-γ, granzyme B, perforin, CXCR3, and CXCR49–13.In scription factors related to iNKT cell differentiation, such as T- addition, Eomes has recently been shown to regulate a specific bet, Gata3, Rorγt as well as the master regulator of all iNKT cells, population of CD8+ T cells in the thymus14. This population, PLZF (Fig. 2a). We also analyzed the expression level of Eomes in which is called IL-4-induced innate memory CD8+ T cells shows iNKT subsets (NKT0, NKT1, NKT2, and NKT17) in the thymus striking upregulation of Eomes but not the related T-box factor, of WT mice. As shown in Fig. 2b, NKT0 express Eomes at higher T-bet14. These findings suggest additional independent roles for levels than other NKT cell subsets. Eomes cKO iNKT cells Eomes in the regulation of particular T cell populations in both expressed higher levels of PLZF and Gata3 protein, but decreased the thymus and peripheral tissues. However the function of levels of T-bet (Fig. 2a–c). Because of the block in the develop- Eomes by itself in iNKT cells remains to be demonstrated. ment of iNKT precursors into NKT1 cells, the frequency of NKT2 To this end, through studies of T cell-specific Eomes condi- and NKT17 subsets tended to increase in the thymus. We then tional knockout (Eomes-cKO) mice, we found that Eomes assessed the phenotypes of iNKT cells in the thymus and spleen, selectively played a role in NKT1 cell development in the thymus. which should be affected by these transcription factors. NK1.1, This impaired development of NKT1 cells in Eomes cKO mice NKG2D, CD69, and CD122, a component of the IL-15 receptor are due to a cell-autonomous effect, as demonstrated by bone that is important for iNKT terminal maturation15, were detected marrow (BM) chimera study. Furthermore, when activated, the in WT mice but their expression was decreased in Eomes cKO remaining iNKT cells in the peripheral tissues in Eomes cKO iNKT cells (Fig. 2d). By contrast, expression of IL-17Rb, which is mice are functionally impaired. The current study reveals the an IL-25 receptor that defines the IL-4 producing capacity of importance of Eomes in the development of NKT1 cells in the iNKT cells21, was substantially increased in Eomes cKO thymus and also in their activation and differentiation in per- iNKT cells (Fig. 2d). ipheral tissues. To better understand Eomes-mediated regulation of the differentiation of iNKT cells, we examined the expression of cytokines, chemokines, and transcription factors in thymic Results iNKT cells by RNA sequencing (RNA-seq). Compared to WT Eomes affects the development of iNKT cells in the thymus.To mice, Eomes cKO mice showed decreased expression of NKT1- investigate the potential role of Eomes in iNKT cell development, related chemokines (Ccl5 and Xcl1), cytokine receptors (Ifnγr1,

2 COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 ARTICLE

a Thymus Spleen Liver 1.20 1.40 14

1.00 1.20 12 1.00 10 0.80 ** ** * 0.80 8 0.60

%iNKT 0.60 6 %iNKT %iNKT 0.40 0.40 4 0.20 0.20 2 0.00 0.00 0 WT Eomes WT Eomes WT Eomes cKO cKO cKO

b Thymus

WT Eomes cKO 104 104

103 0.66103 0.43

102 102 TCRb-PB 101 101

101 102 103 104 101 102 103 104 CD1d/Gal-tetramer-PE

4 10 104 104

3 3 10 10 103

2 2 10 10 102

101 Stage 1 CD44-FITC 10 101 4.78 6.42 0 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD24-APC

104 104 104 11.0 85.6 44.0 40.9 Stage Stage 3 3 3 10 10 10 2 3

102 102 102 Stage 101 1 1 CD44-FITC 10 2.51 10 13.9 1 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 NK1.1-PE/Cy7 c 100 ***

80 ** 60 ** 40

Percentage n.s. 20

0 WT Eomes WT Eomes WT Eomes WT Eomes cKO cKO cKO cKO Stage 0 Stage 1 Stage 2 Stage 3

d e Eomes Thymus Spleen 0.08 900 800 0.07 800 700 ** 700 0.06 ** 600 ** ** 600 ** 500 0.05 500 400 0.04 400 300 0.03

300 NKT-TCR MFI NKT-TCR MFI 200 200 0.02 Relative expression 100 100 0.01 0 0 WT Eomes WT Eomes 0 cKO cKO

Stage 0 Stage 1 Stage 2 Stage 3

Fig. 1 Crucial role of Eomes for iNKT cell terminal differentiation. a Percentage of CD1d-tet+TCRβ+iNKT cells in WT and Eomes cKO thymus, spleen, and liver (n = 6–8, mean ± SEM). b CD24, NK1.1, and CD44 staining on gated iNKT cells from (a). c Percentage of stage 0 to 3 iNKT cells from WT and Eomes cKO mice (n = 6–8, mean ± SEM). d Expression of Vα14TCR in WT and Eomes cKO mice was assessed by ﬂow cytometry as mean ﬂuorescence intensity (MFI). Similar data were obtained from at least three independent experiments. *p < 0.05, **p < 0.01, ***< p < 0.001, Mann–Whitney e Quantitative PCR analysis of Eomes mRNA in thymic iNKT cells (TCRβ+CD1d-tetramer+) from WT mice: stage 0 (CD24+CD44−NK1.1−CD69+), stage 1 (CD24−CD44 −NK1.1−), stage 2 (CD24−CD44+NK1.1−), stage 3 (CD24−CD44+NK1.1+). (n = 6, mean ± SEM) **p < 0.01, two-tailed Student’s t-test

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3

Thymus abWT Eomesc KO c 104 104 Thymus Eomes NKT1 NKT2 NKT17 103 103 0.040 100 100 30

102 102 ** * * 0.035 80 80 101 101 ** TCRb-FITC 0.030 ** ** 20 100 100 60 60 100 101 102 103 104 100 101 102 103 104 CD1d-dimerix/Gal-APC 0.025

4 4 40 40 10 10 % in iNKT % in iNKT 10

3 3 0.020 10 10 20 20 102 102 0.015 NKT17 1 1 0 0 0 Relative expression Relative PLZF-PE 10 10 WT Eomes WT Eomes WT Eomes 0.010 100 100 cKO cKO cKO 100 101 102 103 104 100 101 102 103 104 RORgt-PB 0.005 Spleen 104 104 NKT1 NKT2 NKT17 0.000 100 **60 **15 ** 103 103 NKT0 NKT1 NKT2 NKT17 NKT2 50 102 102 80

1 1 40 10

PLZF-PE 10 10 60 100 100 30 100 101 102 103 104 100 101 102 103 104 GATA3-PE/Cy7 40 % in iNKT % in iNKT 20 % in iNKT5 % in iNKT 104 104 20 103 103 10

2 2 10 10 0 0 0

1 1 WT Eomes WT Eomes WT Eomes

PLZF-PE 10 10 NKT1 cKO cKO cKO 100 100 100 101 102 103 104 100 101 102 103 104 T-bet-PE/Cy7

d 100 100 100 100 100 100 100 80 80 80 80 80 80 80 Thymus 60 60 60 60 60 60 60 40 40 40 40 40 40 40 % of max % of max % of max % of max % of max % of max % of max % of WT 20 20 20 20 20 20 20

0 0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Eomes

100 100 100 100 100 100 100 cKO

80 80 80 80 80 80 80 Isotype Spleen 60 60 60 60 60 60 60 40 40 40 40 40 40 40 % of max % of max % of max % of max % of max % of max % of max % of

20 20 20 20 20 20 20

0 0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD69-PE CD11a-FITC NKG2d-PE CD43-FITC NK1.1-PE CD122-FITC IL17Rb-PE

efCcl5 Xcl1 Ifngr1 Il12rb2 Fcr1γ Il4 Irf4 500 600 200 30 160 80 8 140 70 7 400 500 25 150 120 60 6 400 20 300 100 50 5 300 100 15 80 40 4 FPKM FPKM 200 60 30 3 200 10 50 40 20 2 100 5 100 20 10 1 0 0 0 0 0 0 0 WT Eomes WT Eomes WT Eomes WT Eomes WT Eomes WT Eomes WT Eomes cKO cKO cKO cKO cKO cKO cKO

16 Il10ra 12 Irf825 Stat42.5 Prdm160 Txk80 Tbx21 14 70 10 20 2.0 50 12 60 8 40 10 15 1.5 50 8 6 30 40

FPKM 10 1.0 6 4 20 30 4 20 2 5 0.5 10 2 10 0 0 0 0.0 0 0 WT Eomes WT Eomes WT Eomes WT Eomes WT Eomes WT Eomes cKO cKO cKO cKO cKO cKO

Fig. 2 Aberrant expression of molecules affected by Eomes in iNKT terminal maturation and effector function. a Expression of PLZF, Rorγt, Gata3, and T-bet by thymic and splenic iNKT from WT and Eomes cKO mice detected by ﬂow cytometry. b Quantitative PCR analysis of Eomes mRNA in thymic iNKT cells (TCRβ+CD1d-tetramer+) from WT mice: NKT0 (CD24+CD44−NK1.1−CD8−), NKT1 1(CD24−NK1.1+CD27+CCR6−), NKT2 (CD24−NK1.1-CD27+CD4+), NKT17 (CD24−CD27−CD4−CCR6+CD103+). (n = 5, mean ± SEM) **p < 0.01, two-tailed Student’s t-test. c The percentage of NKT1 (PLZFdimT-bet+), NKT2(PLZFhighGata3+), and NKT17(PLZF+Rorγt+) subsets in thymus and spleen of WT and Eomes cKO mice. (n = 5, mean ± SEM). *p < 0.05, **p < 0.01, Mann–Whitney. d Representative histograms showing expression of CD69, CD11a, NKG2d, CD43, NK1.1, CD122, and IL17Rb by thymic and splenic iNKT cells from WT and Eomes cKO mice (n = 5). Similar data were obtained from at least three independent experiments. e, f Thymic iNKT cells from WT and Eomes cKO mice were analyzed by RNA-Seq. Expression levels (expressed as FPKM) of selected NKT1 (e) or NKT2 (f) subset-speciﬁc genes of thymic iNKT cells were compared between WT and Eomes cKO mice (n = 3, mean ± SEM). (WT vs KO, *p < 0.05, **p < 0.01, ***< p < 0.001, two-tailed Student’s t-test)

Il12rb2, Il10ra), signaling adaptor molecules (FcɛR1γ) and Eomes alters IFN-γ production in iNKT cells. The presence of transcription factors and signaling pathway molecules (Irf8, iNKT cells in Eomes cKO mice allowed us to examine how Eomes Stat4, Prdm1, Txk and Tbx21), but increased expression of deﬁciency may affect iNKT cell effector function. NKT1 cells can NKT2-related molecules such as Il4 and Irf4 (Fig. 2e, f). These produce IFN-γ and IL-4, whereas NKT2 cells produce IL-4 but results indicated that Eomes regulates not only the differentiation, not IFN-γ. NKT17 cells secrete IL-17, but not IFN-γ. Following but also the function of NKT1 cells in the thymus. in vitro stimulation with PMA plus ionomycin for 6 h, WT

4 COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 ARTICLE

a Thymus b NKT1 NKT2 NKT17 PMA/Iono– WT Eomes cKO WT Eomes cKO %IFN-γ + %IL-4+IFN-γ– %IL-17+IFN-γ– 104 104 104 104 iNKT cells iNKT cells iNKT cells 103 0.47 0.0 103 2.33 0.50 103 0.0 0.0 103 0.0 0.0 80 60 20 102 102 102 102 70 50 1 1 1 1 IL-4-PE 10 10 10 10 IL-17-PE 60 ** 15 0 1.94 0 3.67 0 1.96 0 8.68 10 10 10 10 40 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 50 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 PMA/Iono+ IFN-γ-PE/Cy7 IFN-γ-PE/Cy7 40 30 10

104 104 104 104 30 20 3 10.7 34.6 3 47.1 2.18 3 0.19 0.04 3 1.60 0.17 ** 10 10 10 10 20 5 10 102 102 102 102 10

101 101 101 101 0 0 0 IL-4-PE IL-17-PE 61.3 WT Eomes WT Eomes WT Eomes 100 35.2 100 0.98 100 100 3.26 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 cKO cKO cKO IFN-γ-PE/Cy7 IFN-γ-PE/Cy7

cdSpleen NKT1 NKT2 NKT17 γ + + γ– + γ– PMA/Iono– WT Eomes cKO WT Eomes cKO %IFN- %IL-4 IFN- %IL-17 IFN- 4 4 104 104 10 10 iNKT cells iNKT cells iNKT cells

1.04 0.44 2.12 0.25 3 0.19 0.0 3 0.0 0.0 103 103 10 10 60 50 25

2 2 102 102 10 10 50 40 20 IL-4-PE 1 1 101 101 10 10 40 2.07 4.61 0 4.47 0 4.94 100 100 10 10 30 ** 15 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 30 γ γ PMA/Iono+ IFN- -PE/Cy7 IFN- -PE/Cy7 20 10 104 104 104 104 20 12.5 35.0 25.3 0.31 1.63 0.16 5.31 0.19 3 3 3 3 ** 10 10 10 10 10 10 5

102 102 102 102 0 0 0 101 101 101 101

IL-4-PE WT Eomes WT Eomes WT Eomes 18.2 0.72 51.9 1.23 100 100 IL-17-PE100 IL-17-PE 100 cKO cKO cKO 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 IFN-γ-PE/Cy7 IFN-γ-PE/Cy7

e f NKT1 NKT2 NKT17 In vivo Gal i.v. %IFN-γ + %IL-4+IFN-γ– %IL-17+IFN-γ– WT naive WT Eomes cKO iNKT cells iNKT cells iNKT cells 104 104 104 45 1.25 0.93 12.1 31.4 10.8 11.8 25 3.0 103 103 103 40 2.5 102 102 102 35 20 IL-4-PE 101 101 101 30 2.0 1.25 9.63 1.95 15 1 2 3 4 1 2 3 4 1 2 3 4 25 10 10 10 10 10 10 10 10 10 10 10 10 1.5 IFN-γ-PE/Cy7 20 10 104 104 104 15 ** 1.0 0.16 0.39 1.60 4.02 2.12 7.53 103 103 103 10 5 0.5 5 102 102 102 0 0 0.0 1 1 1 IL-17-PE 10 10 10 Naive WT Eomes Naive WT Eomes Naive WT Eomes 2.99 28.2 5.05 101 102 103 104 101 102 103 104 101 102 103 104 cKO cKO cKO IFN-γ-PE/Cy7

Fig. 3 Suppression of the differentiation of IFN-γ producing iNKT cells in Eomes cKO. a, b Percentage of IFN-γ, IL-4, and IL-17A production by thymic iNKT cells stimulated with PMA and Ionomycin (Iono) in WT and Eomes cKO mice. (n = 6–7, mean ± SEM) c, d As in (a, b), percentage of splenic iNKT cells positive for the indicated cytokines in WT and Eomes cKO mice. (n = 6–7, mean ± SEM) e, f Percentage of splenic iNKT cells positive for IFN-γ, IL-4, and IL-17A production in WT or Eomes cKO 2 h after i.v. administration of α-GalCer (1 μg/mouse) (n = 5–6; data are shown as mean ± SEM). Similar data were obtained from at least three independent experiments. **p < 0.01, Mann–Whitney iNKT cells predominantly produced IFN-γ and IL-4, but mini- in NKT2 and NKT17 cells is caused by the decrease of mally produced IL-17 (Fig. 3a, b). In contrast, there was a severe NKT1 cells. reduction in NKT1 cells capable of producing both IFN-γ and IL- Next, we examined the in vivo response of iNKT cells in 4 in the Eomes cKO, while the frequency of NKT2 cells producing Eomes cKO mice. The mice were injected i.v. with α-GalCer only IL-4 increased dramatically (Fig. 3a, b). Similar to thymo- and splenic iNKT cells were analyzed by intracellular cytokine cytes, there were fewer iNKT cells in Eomes cKO spleen that staining 2 h later. Consistent with the in vitro observations, produced both IFN-γ and IL-4 than in WT controls (Fig. 3c, d). fewer Eomes cKO iNKT cells produced both IFN-γ and IL-4 Compared to NKT1 cells, NKT17 cells appeared to increase in than WT iNKT cells (Fig. 3e, f). Together, the regulation of IL-4 Eomes-deﬁcient mice (Fig. 3b–d). These data might suggest that and IFN-γ by Eomes in iNKT cells was developmentally NKT2 and NKT17 cells are selectively increased in Eomes cKO programmed, i.e., the differentiation of NKT1, but not NKT2 mice, but that is not actually the case. The observed increase and NKT17 cells was blocked by Eomes deﬁciency. These

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 5 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3

a Donor CD90.2+

WT Recipient (CD45.1–CD45.2+) BM cells

Eomes cKO (CD45.1+CD45.2+) CD90.1+CD45.2+ lethally irradiated

– b CD90.1 DN DP CD4SP CD8SP 104

3 Eomes cKO 3 3 3 10 10 10 10 103

2.08 86.3 102 102 102 102 102 55.7 64.2 64.2 58.7 101 101 101 101 1 CD8-APC 10 WT 100 100 100 100 CD45.1-BV605 43.3 35.7 35.7 41.2 100 2.47 8.20 3 3 3 3 3 100 101 102 10 104 100 101 102 10 104 100 101 102 10 104 100 101 102 10 104 100 101 102 10 104 CD4-PE CD45.2-FITC

c 100

40 Percentage 20

0 WT Eomes WT Eomes WT Eomes WT Eomes cKO cKO cKO cKO DN DP CD4SP CD8SP

d CD90.1–CD1d-tetramer+TCRb+ Thymus Spleen Liver Eomes cKO 103 103 103 28.4 33.5 14.2 102 102 102

101 101 101 WT 100 66.6 100 58.2 100 82.0 CD45.1-BUV605

3 3 3 100 101 102 10 104 100 101 102 10 104 100 101 102 10 104 CD45.2-FITC

e Thymus Spleen 100 Liver 100 100

80 80 80

60 60 60

40 40 40 Percentage Percentage ** Percentage 20 ** 20 20 **

0 0 0 wt Eomes wt Eomes wt Eomes cKO cKO cKO

f Stage 0 Stage 2 Stage 3 Stage 4

3 3 3 3 10 55.6 10 61.4 10 55.2 10 20.8

102 102 102 102 CD45.1 101 101 101 101

100 37.0 100 22.8 100 43.1 100 75.0

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD45.2

g 100

40 Percentage 20 * 0 WT Eomes WT Eomes WT Eomes WT Eomes cKO cKO cKO cKO Stage 0 Stage 1 Stage 2 Stage 3 observations are consistent with a block at stage 3 in Eomes iNKT cells in a cell-intrinsic manner, we reconstituted lethally cKO mice. irradiated CD45.2+CD90.1+ wild type recipient mice with WT CD45.2+CD90.2+ BM cells and Eomes cKO CD45.1+CD45.2+ CD90.2+ BM cells at a ratio of 1:1 (Fig. 4a). After 8 weeks Cell-intrinsic mechanism in iNKT development by Eomes.To engraftment, CD4−CD8− double negative, double-positive and address whether Eomes regulated the thymic differentiation of single positive thymocytes developed in the recipient mice

6 COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 ARTICLE

Fig. 4 Regulation of NKT1 cell development by Eomes is cell-intrinsic. a Experimental set-up of the BM chimera experiment. CD90.1+CD45.1−CD45.2+ recipient B6.PL mice were lethally irradiated and transplanted i.v. with a 1:1 mixture of CD90.2+ CD45.1−CD45.2+ B6 BM together with BM from CD90.2 +CD45.1+CD45.2+Eomes-cKO mice. Recipient mice were sacriﬁced 8 w after transplantation and iNKT cells in thymus, spleen, liver, and lung were analyzed for the expression of CD45.1 and CD45.2. b Representative dot plots show CD4 and CD8 expression by thymocytes (left) and the indicated subsets (double negative, double-positive and single positive) of chimeric mice (right). c The percentage of each subset generated from WT and KO BM cells. (n = 4, mean ± SEM). d Representative dot plots show CD45.1 and CD45.2 expression by iNKT cells in the thymus, spleen, liver, and lung of the chimeric mice. e The percentage of iNKT cells in each organ generated from WT and KO BM cells. (n = 4, mean ± SEM). f Representative dot plots show CD45.1 and CD45.2 expression by iNKT cells at different development stages (Stage 0–3) within the thymus of the chimeric mice (upper). g The percentage of iNKT cells at each stage generated from WT and KO BM cells. (n = 4, mean ± SEM). Data are combined from two independent experiments. *p < 0.05, **p < 0.01, Mann–Whitney

a Eomes b 0.80 0.70 0 h 48 h 100 100 0.60 80 80 0.50 * 60 60 0.40 Eomes Isotype 0.30 40 40 0.20 20 20 Relative expression 0.10 0 0 –103 0–10103 104 105 3 0 103 104 105 0.00 Eomes-PE Eomes-PE 0 h 16 h 48 h 0 h 16 h WT Eomes cKO

c 0.45 IP:H3K9ac 0.16 IP:H3K27ac 0.40 *** 0.14 0.35 0.12 0.30 * 0.10 *

0.25 % input

0.08 0.20 **

% input ** % input 0.06 0.15 0.10 0.04 0.05 0.02 0.00 0.00 Before After Before After Before After Before After Before After Before After Eomes promoter Eomes TSS Eomes TSS Eomes promoter Eomes TSS Eomes TSS –0.6 kb +1.6 kb –0.6 kb +1.6 kb

Fig. 5 Transient expression of Eomes by iNKT cells is epigenetically regulated. a Kinetics of Eomes mRNA expression in non-activated (0 h) and activated (16, 48 h) thymic iNKT cells. Total thymic iNKT cells from WT mice were stimulated with anti-CD3 plus anti-CD28 mAbs for the indicated periods and the levels of Eomes transcripts were determined by qPCR. Sorted thymic iNKT cells from Eomes cKO were used as a negative control. (n = 3–7, mean ± SEM). Data are combined from four independent experiments. b Expression of Eomes in non-activated (0 h) and activated (48 h) thymic iNKT cells assessed by ﬂow cytometry. (Red, Eomes; Black, isotype) (n = 3) c Chip analysis for H3K9ac and H3K27ac modiﬁcations at the Eomes locus in non-activated and activated thymic iNKT cells (n = 4, mean ± SEM). Data are summarized from three independent experiments. *p < 0.05, **p < 0.01, ***< p < 0.001, Mann–Whitney

(Fig. 4b). The WT to KO ratios of double negative, double- Eomes-deficient stage 3 iNKT cells (Fig. 4f, g). These findings positive and single positive thymocytes ranged from 0.8–1.2, close suggested an essential and cell-intrinsic role for Eomes in the to the original 1:1 ratio (Fig. 4b, c), suggesting that Eomes is not terminal maturation and maintenance of iNKT cells in the essential for αβT cell maturation in the thymus, even in a com- thymus. petitive environment. We verified that iNKT cells were also reconstituted in the mixed chimeric mice (Fig. 4d–g), and compared the ability of each donor population to reconstitute the TCR signaling induces Eomes expression in thymic iNKT cells. + CD1d-Tet iNKT cells in the thymus, spleen, liver, and lung. As shown in Fig. 1e, the expression of Eomes in iNKT cells in the When we focused on the reconstituted iNKT cells in the thymus, steady state is quite low. Next, we examined whether Eomes in we found fewer iNKT cells of Eomes cKO origin than of wild type iNKT cells can be upregulated by TCR stimulation. For this, origin, the WT to KO ratios of iNKT cells were more than 2 iNKT cells were sorted from thymus and stimulated with anti- (Fig. 4d, e). Similar results were also observed with splenocytes CD3 and anti-CD28 mAbs. We found that the expression of and liver mononuclear cells (MNCs) from the chimeric mice Eomes mRNA was upregulated at 16 h after TCR stimulation, but (Fig. 4d, e) Furthermore, we analyzed the stages of iNKT cells and not in Eomes cKO mice (Fig. 5a) and was also elevated at the found a similar frequency of Eomes-deficient and wild-type protein level 48 h after the stimulation (Fig. 5b). These results derived Stage 0-Stage 2 cells, but a greatly decreased frequency of indicate that expression of Eomes can be induced upon TCR

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 7 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3

DC/Gal immunized a WT b e DC/Gal immunized naive WT Eomes cKO WT 104 104 104 80 naive WT Eomes cKO * 100 100 100 103 103 103 70 80 80 80

102 102 102 60 CD49d-PE 60 60 60 % of max % of max % of max 0.21 0.78 0.57 40 40 40 101 101 101 50 20 20 20 iNKT 0 0 0 0 0 0 + 10 10 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 40 PE-A: CD49d PE-A PE-A: CD49d PE-A PE-A: CD49d PE-A 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 100 100 CD1d-dimerix/Gal-APC 30 80 80 80 104 104 104 % Klrg1 20 NKG2D-PE 60 60 60 % of max % of max % of max 103 103 103 40 40 40 10 20 20 20 2 2 2 10 10 10 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 92.5 6.43 0 PE-A: CD69 PE-A PE-A: CD69 PE-A PE-A: CD69 PE-A 1 96.9 3.07 1 30.5 68.7 1 Gal-APC10 CD19-PB 10 10 WT WT Eomes cKO 100 100 100 CD1d-dimerix/ 0 0 0 naive 80 80 80 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 DC/Gal immunized 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 NK1.1-PE 60 60 60 % of max % of max % of max Klrg1-PE/Cy7 40 40 40 c d 20 20 20 60 * 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 DC/Gal immunized PE-A: NK11 PE-A PE-A: NK11 PE-A PE-A: NK11 PE-A WT 50 100 100 100 naive WT Eomes cKO 80 80 80 104 104 104 Ly6C-FITC 60 60 60 3 3 3 40 % of max % of max % of max 10 10 10 40 40 40

iNKT 20 20 20 2 2 2 + 10 10 10 30 0.37 56.7 0.32 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 1 1 1 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FITC-A: Ly6C FITC-A FITC-A: Ly6C FITC-A FITC-A: Ly6C FITC-A GzmA-PE 100 100 100 0 0 0 20

10 10 10 % GzmA 80 80 80 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Klrg1-PE/Cy7 CD69-PE 60 60 60 10 % of max % of max % of max 40 40 40

20 20 20

0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 WT WT Eomes cKO PE-A: NKG2D PE-A PE-A: NKG2D PE-A PE-A: NKG2D PE-A naive DC/Gal immunized

Fig. 6 Eomes-deficient iNKT cells fail to differentiate into Klrg1+ iNKT cells after DC/Gal immunization. WT and Eomes cKO mice were immunized with DC/Gal. Four weeks later, lung iNKT cells were analyzed by flow cytometry. a, b The expression and frequency of lung Klrg1+ iNKT cells from naïve WT or DC/Gal-immunized WT or Eomes cKO mice. (n = 5) c, d The expression and frequency of lung granzyme A+ iNKT cells (n = 5, mean ± SEM) e Expression of the indicated proteins by lung iNKT cells from WT naïve mice or DC/Gal-immunized, WT or Eomes cKO mice. (n = 5) Similar data were obtained from at least three independent experiments. *p < 0.05, Mann–Whitney stimulation of iNKT cells. Thus, Eomes shows a unique expres- To further characterize the gene-expression program in iNKT sion pattern, with little expressed in the steady state. It is cell subsets in the lung from WT and Eomes cKO mice given DC/ expressed transiently, but apparently only in the early activation Gal, we performed pairwise comparison of bulk RNA-seq (Figs 7 phase. We hypothesized that such transient expression should be and 8). In the steady state, we found no difference in iNKT cells regulated epigenetically and therefore evaluated histone acetyla- from WT and Eomes cKO mice. Given that NKT2 and NKT17 tion (ac), an epigenetic modification associated with accessible are dominant in the lung in the steady state22, this result was chromatin structure and active transcription. As shown in Fig. 5c, predictable. In contrast, we observed obvious differences in both H3K9ac and H3K27ac were increased at the Eomes locus in function-related molecules, such as effector molecules, cytokines, activated iNKT cells. and chemokines, by the transcriptome analysis of activated iNKT cells of Eomes cKO and WT mice after immunization with DC/Gal (Fig. 7). Transcripts encoding NKT1 effector molecules, Eomes regulates the activation of iNKT cells in periphery. After e.g., GzmA, GzmB, and Fasl, were more highly expressed in iNKT showing that Eomes regulates the differentiation and function of from WT mice injected with DC/Gal than in untreated or DC/ NKT1 cells in the thymus, we next examined its effect on the Gal-injected Eomes cKO mice (Fig. 8a). The iNKT cells in DC/ activation phase of iNKT cells in peripheral tissues, which was Gal-injected Eomes cKO mice showed high level expression of unknown. Lee et al. reported that NKT2 and NKT17 cells pre- transcripts encoding NKT2 or NKT17 type cytokines and dominate in lung in the steady state22. However, we previously chemokines (Ccr2, Ccr4, Il17rb, Il13, Il17a) and signaling reported that the number of NKT1-polarized Klrg1+ iNKT cells pathway molecules (Gata3, Irf4, Zbtb16, Ikzf3, Rorc, and Nr1d1) markedly increased in the lung of WT mice after vaccination with (Fig. 8b, c). Thus, when DC/Gal was administered, NKT1 cells DC/Gal7. We also observed dominant expression of Eomes in were increased in WT mice, whereas NKT2 and NKT17 cells were Klrg1+ iNKT cells, but not in naïve iNKT cells. As previously increased in Eomes cKO mice. The NKT1 transcript signatures demonstrated, we verified the expression of Klrg1 and granzyme are therefore reciprocally regulated by Eomes with transcripts of A (Fig. 6a–d) as well as NK1.1, CD49d, NKG2D, Ly6C, and CD69 NKT2 and NKT17 cells during their activation in peripheral (Fig. 6e) by WT Klrg1+ iNKT cells in the lung after DC/Gal organs. immunization. By contrast, in the DC/Gal-injected Eomes cKO + + mice, the generation of Klrg1 gzmA lung iNKT cells was Discussion inhibited (Fig. 6a–d), as was the expression of CD49d, NKG2D, Eomes is well known to regulate the differentiation of certain NK1.1 and Ly6C (Fig. 6e). These results indicate that the per- effector cells, such as CD4+ and CD8+ T cells and NK cells, and ipheral differentiation of iNKT cells triggered by iNKT ligand and is also involved in memory T cell induction23. In the current TCR signaling is also regulated by Eomes. And, they also suggest study, we demonstrated an impact of Eomes on iNKT differ- that NKT1 cells may require TCR signaling during their differentiation in the thymus and on the activation program in per- entiation into memory-like iNKT cells. ipheral iNKT cells. Eomes controls the differentiation from the

8 COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 ARTICLE

WT lung NKT –5.0 1:1 5.0 EomescKO lung NKT

S09482 S09483 S09484 S09485 S09486 S09487 S09489 S09488 S09490 S09491 S09492 S09493 WT DCG-lung NKT

EomescKO DCG-lung NKT

Ifng Cxcr3 Ccl4 Klra1 Klrb1c Arsb Stard10 Fasl Serpinb9 Spry2 KIrk1 Itgb1 Gzmb Ms4a4b Klrg1 Serpinb6b Epsti1 Ly6c2 Nkg7 Pdcd1 Dusp4 Tmem176b II17rb Ramp1 1300014I06Rik Tmem176a Gpr183 Nt5e Znrf1 II7r Capg Emb Lmo4 Ccr8 Atf3 Smox Furin Cmklr1 Lair1 Entpd1 Cx3cr1 Tm6sf1 Stk32c Baiap3 Soat2 As3mt Plcg2 Kcnj8 S1pr5 Gpx8 Gzmk Nod1 Slamf7 Itga1 Gas7 Ablim3 Pcsk1 II17re II23r 9430020K01Rik Sdc4 Tox2 II1rl1 Dkk3 Tiam1 Mtss1 Ptpn13 Lif Vdr Hlf Pparg Chdh Gtf2ird1 Coro2b Mmp25 Sdc1 II17a Rore lI5 St6gal1 Areg Tox D18Ertd653e Lrig1 Spry1 Nav2 3110043O21Rik Ltb4r1 Myo1e Cpm Egln3 Ndrg1 II2ra Itgae Ifngr2 Cd83 Nr1d1 Sh3bp5 Tnfsf11 II13

Fig. 7 Differentially expressed genes between iNKT cells from naive or DC/Gal primed WT and Eomes cKO mice. WT and Eomes cKO mice were immunized i.v. with DC/Gal. One week later, lung iNKT cells were sorted and analyzed by RNA-Seq. The differential expression analysis was performed using an R based package, EdgeR with the raw counts from RNA-seq data of lung iNKT cells from DC/Gal primed WT and Eomes cKO mice (n = 3). Clustering data of top 100 differentially expressed genes are shown in a heat map format with their gene symbols

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 9 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3

a Ccl4 Ccl5 Fasl Gzma Gzmb 200 14,000 140 6000 800 12,000 120 5000 150 600 10,000 100 4000 8000 80 100 3000 400 6000 60 FPKM 2000 50 4000 40 200 * 2000 ** 20 * 1000 *** ** 0 0 0 0 0 DCG DCG DCG DCG DCG DCG DCG DCG DCG DCG Naive Naive Naive Naive Naive Naive Naive Naive Naive Naive WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO

400 Ifng400 Klrg1150 Tbx2160 Irf8100 Runx3 50 300 300 80 100 40 60 200 200 30 **

FPKM 40 50 20 ** 100 100 ** ** ** 10 20 0 0 0 0 0 DCG DCG DCG DCG DCG DCG DCG DCG DCG DCG Naive Naive Naive Naive Naive Naive Naive Naive Naive Naive

WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO

b Il4 Ccr4 Il17rb Il13 Gata3 100 70 *** 140 ** 200 150 ** 60 120 * 80 150 50 100 100 60 40 80 100 30 60

FPKM 40 50 20 40 50 20 10 20 0 0 0 0 0 DCG DCG DCG DCG DCG DCG DCG DCG DCG DCG Naive Naive Naive Naive Naive Naive Naive Naive Naive Naive

WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO

50 Irf470 Zbtb16 ** 60 * 40 50 30 40 30

FPKM 20 20 10 10 0 0 DCG DCG DCG DCG Naive Naive Naive Naive

WT KO WT KO WT KO WT KO

c 100 Ccr2350 Il17a** 80 Itgae * 150 Ikzf3** 100 Rorc ** 300 * 80 60 80 250 100 60 200 60 40 150 40 FPKM 40 50 100 20 20 50 20 0 0 0 0 0 DCG DCG DCG DCG DCG DCG DCG DCG DCG DCG Naive Naive Naive Naive Naive Naive Naive Naive Naive Naive

WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO WT KO

30 Nr1d1 ** 25 20 15

FPKM 10 5 0 DCG DCG Naive Naive

WT KO WT KO

Fig. 8 Comparison of RNA proﬁles of lung iNKT cells from naive or DC/Gal primed WT and Eomes cKO mice. WT and Eomes cKO mice were immunized i.v. with DC/Gal. One week later, lung iNKT cells were sorted and analyzed by RNA-Seq. a–c Expression levels (expressed as FPKM) from selected NKT1 (a) or NKT2 (b) or NKT17 (c) subset-speciﬁc genes of the lung iNKT cells between naïve or DC/Gal-primed WT and Eomes cKO mice were compared. (n = 3, mean ± SEM). (WT DC/Gal vs cKO DC/Gal, *p < 0.05, **p < 0.01, ***< p < 0.001, two-tailed Student’s t-test)

10 COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 ARTICLE iNKT precursor to NKT1 cells in the thymus. In addition, Eomes a also regulates NKT1 cell differentiation in the peripheral tissues. Thymus We previously showed that after activation with the iNKT ligand- iNKT precursor loaded DCs in the peripheral tissues, some activated iNKT cells Eomes ↑ become skewed toward long-term memory type NKT1 cells. However, they fail to do so in the Eomes cKO mice. Three recent studies analyzed global gene expression profiles of thymic NKT1, NKT2, and NKT17 cells and their enhancer landscape and revealed that the three subsets are transcriptionally distinct24–26. Engel et al. identified the enhancer profiles of each NKT subset by H3K27ac Chip-Seq25. They found that enhancers in NKT1, NKT2, and NKT17 cells that had increased H3K27ac showed enrichment at binding motifs for T-bet, Gata family and Rorγt, respectively. With regard to Eomes, they described NKT1 NKT2 NKT17 enrichment at Eomes binding motifs in the enhancers in T-bet Gata3 Rorgt NKT1 cells. However, because expression of Eomes in iNKT cells PLZFlo PLZFhi PLZFhi was low, they suspected that this finding might reflect the activity IFN-γ IL-4 IL-17 of another transcription factor with a similar motif25. It is true IL-4 IL-13 IL-22 that Eomes expression is quite low in iNKT cells in the steady Ccl5 IL-17Rb IL-23R state (Figs 1, 5). Some studies previously proposed that stage 0 b iNKT cells received a stronger TCR signal during selection rather CD1d α-GalCer than mature iNKT cells using Nur77GFP mice, which upregulate Lung GFP in response to Ag receptor but not inflammatory signals16,17. NKT1 Indeed, we showed that among thymic iNKT cells, the highest expression of Eomes was in stage 0 iNKT cells and then it – Eomes ↑ decreased in stage 1 3 iNKT cells. We also showed that upre- DC gulation of Eomes in the thymic iNKT cells was induced by TCR stimulation in vitro (Fig. 5). Both of these findings suggest that Eomes upregulation during iNKT cell development in thymus may be induced by TCR signaling. Klrg1+ iNKT1 The relationship between Eomes expression and the acquisition of NKT1 cell phenotype and function during the development of T-bet iNKT cells in the thymus is unclear. It is known that different Runx NKT cell subsets express different levels of TCR26,27. In addition IFN-γ to such TCR signal strength, transcription factors, epigenetic gzmA, gzmB changes, and cytokines may play a role in the development of Ccl3, Ccl4, Ccl5 NKG2Dhi iNKT subsets in the thymus. CD122 is a shared subunit of IL-2 CD49dhi and IL-15 receptors, and responsiveness to IL-15 is essential for the final maturation into Stage 3 iNKT cells6. Also, after receiving Fig. 9 The model depicting the role of Eomes during iNKT development in a signal via CD122-IL-15, the micro-RNA let-7 is upregulated in the thymus (a) and differentiation in the lung (b) (see Discussion for iNKT precursor cells and that inhibits PLZF, thus skewing NKT1 details) differentiation28. It thus seems possible that IL-15-CD122 may help in the transit from iNKT cell precursor toward NKT1 differentiation. By transcriptome analysis of thymic iNKT cells from within the lung parenchyma, whereas NKT1 cells were detected WT and Eomes cKO mice, we found that expression of NKT1- in both vasculature and parenchyma22. Intravenously adminis- fi speci c molecules, i.e., cytokine and chemokine receptors as well tered DC/Gal can be trapped and retained in the lung for at least as cytokines, chemokines and transcription factors, was 24 h7. Therefore, these intravascular iNKT cells in the lung could decreased, whereas NKT2- and NKT17-related molecules were interact directly with DC/Gal, thereby receiving TCR as well as 6,26,27,29 upregulated (Fig. 2e, f) . These may be affected by the cytokine signaling. In the current study, we demonstrated that altered ratio of NKT1, 2 and 17 cells. However, it was previously Klrg1+ iNKT cells are not generated in Eomes cKO mice, even reported that CD122 expression is a direct target of Eomes in after administration of DC/Gal. These results clearly imply that 9 T cells . There thus remains the possibility of direct regulation of generation of Klrg1+ iNKT cells requires Eomes expression CD122 by Eomes in iNKT cells. In elucidating an interaction (Fig. 9). Given that Klrg1+ iNKT cells express Eomes, TCR sig- between Eomes and CD122 in the development of thymic naling and cytokine signaling may reactivate Eomes expression NKT1 cells, further studies of whether it interacts directly or and switch the peripheral differentiation of NKT1 cells into the indirectly through other cytokines or transcription factors will be Klrg1+ iNKT cell pathway. Taken together, Eomes is crucial, not required. + only for the development of iNKT cells in the thymus, but also for Klrg1 iNKT cells in the lung were generated and maintained their further differentiation in the periphery. for the long term after administration of DC/Gal (Fig. 9)7. These cells displayed NKT1-like cytokine and chemokine profiles (predominant IFN-γ, CCL3, -4, and -5 production) and pro- Methods longed survival as memory-like iNKT cells expressing Tbx21, Mice. C57BL/6 mice and B6.PL were purchased from CLEA Japan and Jackson + Runx3, and Eomes. The molecular mechanism by which the Laboratory respectively. EomesExon1-f/Exon1-f-CD4Cre mice (Eomes cKO) were Klrg1+ iNKT population is generated after administration of DC/ kindly provided by Dr. T. Nakayama (Chiba University) and Dr. Steven L. Reiner fi (Columbia University) and backcrossed to C57BL/6 mice for 12 generations. Mice Gal is not yet clearly de ned. As previously reported, NKT2 cells 6–8 wk of age were used for experiments. All the mice were maintained under localize inside the blood vasculature and NKT17 cells reside specific pathogen-free conditions and all procedures were performed in compliance

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 11 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 with the protocols approved by the Institutional Animal Care Committee at sequencing was carried out using a HiSeq1500 equipment (Illumina). The sequence RIKEN. Mixed-bone marrow (BM) chimeras containing wild-type (CD45.1 reads were mapped to the mouse genome (NCBI version 37) using TopHat2 −CD45.2+CD90.2+) and Eomes cKO BM (CD45.1+CD45.2+CD90.2+) at a ratio (version 2.0.8) and botwie2 (version 2.1.0) with default parameters, and gene of 1:1 were generated by lethal irradiation of B6.PL recipients (CD45.1−CD45.2 annotation was provided by NCBI. Transcript abundances were estimated using ﬂ ﬂ +CD90.2+) (two doses of 5 Gy each), followed by reconstitution by intravenous Cuf inks (version 2.1.1). Cuf inks was run with the same reference annotation injection of 1 × 107 donor BM cells. Mixed BM transfer mice were analyzed 8 weeks with TopHat2 to generate FPKM (fragments per kilobase per million mapped after transplantation. reads) values for known gene models.

Cell preparation. Thymocytes and splenocytes were obtained by pressing thy- Chromatin immunoprecipitation (ChIP) and ChIP-qPCR.ChIPwasaspre- 31 fi mus and spleen through a 70 μm cell strainer and erythrocytes were lysed with viously described .SortediNKTcellswere xed with 1% formaldehyde for ACK lysing buffer (GIBCO) followed by two washes in RPMI. For isolation of 10 min at RT, followed by quenching of formaldehyde with 125 mM glycine for 5 min at RT. Fixed iNKT cells were frozenbyliquidnitrogen,andstoredin lung and liver MNCs, the tissues were digested with collagenase D (Roche) and – then layered on Percoll gradients (40/60%) (Amersham Pharmacia Biotec) and 80 °C. Cross-linked genomic DNA was lysed in lysis buffer [50 mM Tris-HCl centrifuged for 20 min at 900 × g. BM-derived DCs were generated in the pre- (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS, and 1 × cOmplete Protease Inhibitor sence of GM-CSF and pulsed with 100 ng ml−1 α-GalCer for 48 h from day 6 and Cocktail (Roche)], and sonicated with a Picoruptor (Diagenode), 15 cycles of 30 sonication (30 s) and interval (30 s) at 4 °C. A complex of Dynabeads Protein G matured by LPS as previously described . For RNA isolation from thymic μ fi μ iNKT cells, thymocytes were enriched for iNKT cells by negative selection using (50 l, ThermoFisher Scienti c) and antibody (5 g) was formed in 0.02% anti-CD8 micro beads (Milteny Biotec) and MACS magnet and LS columns. The Tween 20 in PBS for 6 h at 4 °C. Then, ChIP was performed in IP buffer [20 mM remaining cells were stained with CD1d-dimers loaded with α-GalCer followed Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0), 1% Triton-X100, 150 mM NaCl, and by anti-Mouse IgG-PE and anti-TCR-β-PB and Lived/Dead Aqua. Then, 1×cOmpleteProteaseInhibitorCocktail]for21hat4°C.ChIPsampleswere TCRβint CD1d-dimer/Gal+ cells (iNKT cells) were sorted with a FACSAria (BD) washed with wash buffer (50 mM HEPES-KOH (pH 7.5), 500 mM LiCl, 1 mM (purity,>95%).ForRNAisolationfromthymiciNKTcellstagesorsubsets, EDTA, 0.7% sodium deoxycholate, 1% Nonidet P-40) 5 times, followed by a wash with TE buffer (Nippongene) twice. DNA was eluted in elution buffer thymus cell suspensions were pooled from 6 WT mice. Each iNKT cell (CD1d- μ tet/Gal+TCRbint)stagewassortedasfollowed:stage0(CD8−CD24+CD44 [50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS, and 40 gPro- − − + − − − − teinase K (Wako)] for 16 h at 65 °C. DNA was purified with MinElute PCR NK1.1 CD69 ), stage 1 (CD24 CD44 NK1.1 ), stage 2 (CD24 CD44 fi + − − + + Puri cation Kit (Qiagen). Antibodies for ChIP were anti-H3K9ac (Millipore, NK1.1 ), stage 3 (CD24 CD44 NK1.1 ) (SupplementaryFig.4a).iNKT – + − #06 599), anti-H3K27ac (Active Motif, #39133), and normal rabbit IgG (Mil- subsets (CD1d-tet/Gal TCRbint) were sorted as followed:25 NKT0 (CD8 CD24 – + − − − + + − − lipore, #12 370). Enrichment was analyzed with qPCR (see above) using the CD44 NK1.1 ), NKT1(CD24 NK1.1 CD27 CCR6 ), NKT2 (CD24 NK1.1 standard curve method. Gene-specific forward and reverse primers are descri- −CD27+CD4+), NKT17 (CD24−CD27−CD4−CCR6+CD103+)(Supplementary bed in Supplementary Table 1. Fig. 4b). For RNA isolation from lung iNKT cells, lung MNCs were stained with CD1d-dimers loaded with α-GalCer, followed by anti-Mouse IgG-PE and anti- − + Statistical analysis. Statistical analysis was performed using StatMate (Ver 5.01). CD19-PB and Lived/Dead Aqua. Then, CD19 CD1d-dimer/Gal cells The F-test was used to tests for normal distribution of the data. Differences were (iNKT cells) were sorted with a FACSAria (BD). analyzed using a two-tailed Student’s t-test or Mann–Whitney U-test. All data are presented as mean ± SEM. p < 0.05 was considered statistically significant. Flow cytometry. The following monoclonal antibodies (mAbs) were purchased from BD Bioscience, BioLegend, or e-Bioscience: anti-CD4 (GK1.5), anti-CD8 Reporting summary. Further information on experimental design is available in (53.67), anti-CD16/32 (93), anti-CD19 (6D5), anti-CD24 (M1/19), anti-CD27 the Nature Research Reporting Summary linked to this article. (LG.3A10), anti-TCRβ (H57-597), anti-CD43 (S7), anti-CD44 (1M7), anti-CD11a (M17/4), anti-CD69 (H1.2F3), anti-NKG2D (CX5), anti-NK1.1 (PK136), anti- CD103 (2E7), anti-CCR6 (29-2L17), anti-CD122 (TM-β1), anti-CD45.1 (A20), Data availability anti-CD45.2 (104), anti-EGR2 (erongr2), anti-T-bet (eBio4B10), anti-Gata3 RNA-seq data that support the findings of this study have been deposited in the Gene (TWAJ), anti-Rorγ (B2D), anti-PLZF (9E12), anti-Eomes (Dan11mag), anti-IFN-γ Expression Omnibus (GEO) database with accession codes GSE128069. Source data (XMG1.2), anti-IL-4 (11B11), anti-IL-17 (TC11-18H10), anti-IL-17RB(9B10) and underlying the graphs presented in the main figures is available in Supplementary Data 1. CD1d-dimers. CD1d-tetramers were purchased from MBL. Fixable Aqua Dead Cell stain kit (Invitrogen) was used to eliminate dead cells. Intracellular staining for transcription factors was performed using the eBioscience Foxp3 Staining Buffer Received: 23 August 2018 Accepted: 18 March 2019 Kit. Intracellular staining for cytokines was performed using the BD Cytofix/ Cytoperm™ Kit. For analysis, a FACSCalibur, Canto II or LSRFortessa X-20 instrument and the CELLQuest or FACSDiva (BD Biosciences) or FlowJo software (v10.3B2) packages were used.

Activation of iNKT cells. For in vivo activation, mice were administered 1 μg α- References GalCer (Funakoshi) by i.v. injection. Two hours later, splenocytes were analyzed 1. D’Cruz, L. M., Yang, C. Y. & Goldrath, A. W. Transcriptional regulation of for cytokine production. For in vitro activation, 5 × 106 splenocytes or thymocytes NKT cell development and homeostasis. Curr. Opin. Immunol. 22, 199–205 were stimulated with 50 ng/ml PMA (SIGMA) and 500 ng/ml ionomycin (SIGMA) (2010). in the presence of GolgiPlug (BD) for 6 h. In some experiments, sorted thymic 2. Szabo, P. A., Anantha, R. V., Shaler, C. R., McCormick, J. K. & Haeryfar, S. M. iNKT cells were stimulated with 10 μg/ml immobilized anti-CD3 mAb and 2 μg/ml CD1d- and MR1-restricted T cells in sepsis. Front. Immunol. 6, 401 (2015). soluble anti-CD28 mAb for 16 h or 48 h. 3. Constantinides, M. G. & Bendelac, A. Transcriptional regulation of the NKT cell lineage. Curr. Opin. Immunol. 25, 161–167 (2013). Quantitative PCR 4. Hu, T., Gimferrer, I. & Alberola-Ila, J. Control of early stages in invariant . Cells were lysed in TRIzol LS (Invitrogen) and the RNA pre- – cipitation was done with 20 μg RNAase-free glycogen (Invitrogen) and 500 μl natural killer T-cell development. Immunology 134,1 7 (2011). Isopropanol (SIGMA). The RNA pellets were washed with 1 ml of 70% cold 5. Martinez, G. J. et al. The transcription factor NFAT promotes exhaustion of + – ethanol. Extracted RNA was quantified with Quant-iT™ RiboGreen® RNA Assay activated CD8 T cells. Immunity 42, 265 278 (2015). Kit (Thermo Fisher Scientific). cDNA synthesis was performed with the Ovation 6. Seiler, M. P. et al. Elevated and sustained expression of the transcription qPCR system (Nugen) from 10 ng RNA as a template. When RNA concentration factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR was lower than 2.5 ng/μg, SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) signaling. Nat. Immunol. 13, 264–271 (2012). was used for cDNA synthesis from 1 ng RNA as a template. 7. Shimizu, K. et al. KLRG+ invariant natural killer T cells are long-lived Quantitative PCR was performed using a StepOne Plus instrument (Applied effectors. Proc. Natl Acad. Sci. USA 111, 12474–12479 (2014). Biosystems) with FastStart Universal Probe Master mix (Roche). The following 8. Kaech, S. M. & Cui, W. Transcriptional control of effector and memory CD8+ primer pair and probes were used: Eomes (forward) 5′-accggcaccaaactgaga-3′, T cell differentiation. Nat. Rev. Immunol. 12, 749–761 (2012). (reverse) 5′-aagctcaagaaaggaaacatgc-3′, UPL probe #9. HPRT1 (forward) 5′- 9. Intlekofer, A. M. et al. Effector and memory CD8+ T cell fate coupled by T- cctcctcagaccgcttttt-3′, (reverse) 5′-aacctggttcatcatcgctaa-3′, UPL probe #95. Relative bet and eomesodermin. Nat. Immunol. 6, 1236–1244 (2005). gene expression was calculated using the ΔΔCT method with the expression of 10. Banerjee, A. et al. Cutting edge: The transcription factor eomesodermin HPRT1 as the internal control. enables CD8+ T cells to compete for the memory cell niche. J. Immunol. 185, 4988–4992 (2010). fl Next-generation sequencing (RNA-seq). RNA was prepared with the RNeasy 11. Joshi, N. S. et al. In ammation directs memory precursor and short-lived + Plus Micro Kit (Qiagen). RNA-seq library samples were prepared using an NEB- effector CD8( ) T cell fates via the graded expression of T-bet transcription – Next Ultra RNA Library Prep Kit for Illumina (New England BioLabs). The factor. Immunity 27, 281 295 (2007).

12 COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0389-3 ARTICLE

12. Pipkin, M. E. et al. Interleukin-2 and inflammation induce distinct 30. Fujii, S., Shimizu, K., Kronenberg, M. & Steinman, R. M. Prolonged transcriptional programs that promote the differentiation of effector cytolytic interferon-g producing NKT response induced with a-galactosylceramide- T cells. Immunity 32,79–90 (2010). loaded dendritic cells. Nat. Immunol. 3, 867–874 (2002). 13. Intlekofer, A. M. et al. Requirement for T-bet in the aberrant differentiation of 31. Murai, F. et al. EZH2 promotes progression of small cell lung cancer unhelped memory CD8+ T cells. J. Exp. Med. 204, 2015–2021 (2007). by suppressing the TGF-β-Smad-ASCL1 pathway. Cell Discov. 1, 15026 14. Renkema, K. R. et al. IL-4 sensitivity shapes the peripheral CD8+ T cell pool (2015). and response to infection. J. Exp. Med. 213, 1319–1329 (2016). 15. Intlekofer, A. M. et al. Anomalous type 17 response to viral infection by CD8 + T cells lacking T-bet and eomesodermin. Science 321, 408–411 (2008). Acknowledgements 16. Moran, A. E. et al. T cell receptor signal strength in Treg and iNKT cell This work was supported by JSPS KAKENHI Grant Number JP26460583. We thank P.D. development demonstrated by a novel fluorescent reporter mouse. J. Exp. Burrows (University of Alabama at Birmingham) for critical reading of the manuscript Med. 208, 1279–1289 (2011). and Ms. Sakurai and Yumoto for technical assistance. 17. Lee, Y. J., Holzapfel, K. L., Zhu, J., Jameson, S. C. & Hogquist, K. A. Steady- state production of IL-4 modulates immunity in mouse strains and is Author contributions determined by lineage diversity of iNKT cells. Nat. Immunol. 14, 1146–1154 K.S. and S.F. designed, supervised most of the experiments and wrote the paper. Y.S., (2013). M.K., H.N., and K.S. performed the experiments. T.W. and O.O. processed RNAseq. 18. Townsend, M. J. et al. T-bet regulates the terminal maturation and homeostasis of NK and Vα14i NKT cells. Immunity 20, 477–494 (2004). 19. Coquet, J. M. et al. Diverse cytokine production by NKT cell subsets and Additional information identification of an IL-17-producing CD4-NK1.1- NKT cell population. Proc. Supplementary information accompanies this paper at https://doi.org/10.1038/s42003- Natl Acad. Sci. USA 105, 11287–11292 (2008). 019-0389-3. 20. Bennstein, S. B. Unraveling natural killer T-cells development. Front. Immunol. 8, 1950 (2017). Competing interests: The authors declare no competing interests. 21. Terashima, A. et al. A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity. J. Exp. Reprints and permission information is available online at http://npg.nature.com/ Med. 205, 2727–2733 (2008). reprintsandpermissions/ 22. Lee, Y. J. et al. Tissue-specific distribution of iNKT Cells impacts their cytokine response. Immunity 43, 566–578 (2015). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in 23. Lazarevic, V., Glimcher, L. H. & Lord, G. M. T-bet: a bridge between innate published maps and institutional affiliations. and adaptive immunity. Nat. Rev. Immunol. 13, 777–789 (2013). 24. Lee, Y. J. et al. Lineage-specific effector signatures of invariant NKT cells are shared amongst γδ T, innate lymphoid, and Th cells. J. Immunol. 197, Open Access This article is licensed under a Creative Commons 1460–1470 (2016). Attribution 4.0 International License, which permits use, sharing, 25. Engel, I. et al. Innate-like functions of natural killer T cell subsets result from adaptation, distribution and reproduction in any medium or format, as long as you give highly divergent gene programs. Nat. Immunol. 17, 728–739 (2016). appropriate credit to the original author(s) and the source, provide a link to the Creative 26. Georgiev, H., Ravens, I., Benarafa, C., Forster, R. & Bernhardt, G. Distinct Commons license, and indicate if changes were made. The images or other third party gene expression patterns correlate with developmental and functional traits of material in this article are included in the article’s Creative Commons license, unless iNKT subsets. Nat. Commun. 7, 13116 (2016). indicated otherwise in a credit line to the material. If material is not included in the 27. Tuttle, K. D. et al. TCR signal strength controls thymic differentiation of iNKT article’s Creative Commons license and your intended use is not permitted by statutory cell subsets. Nat. Commun. 9, 2650 (2018). regulation or exceeds the permitted use, you will need to obtain permission directly from fi 28. Pobezinsky, L. A. et al. Let-7 microRNAs target the lineage-speci c the copyright holder. To view a copy of this license, visit http://creativecommons.org/ transcription factor PLZF to regulate terminal NKT cell differentiation and licenses/by/4.0/. effector function. Nat. Immunol. 16, 517–524 (2015). 29. Zhao, M. et al. Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70. Nat. Commun. 9, 2627 (2018). © The Author(s) 2019

COMMUNICATIONS BIOLOGY | (2019) 2:150 | https://doi.org/10.1038/s42003-019-0389-3 | www.nature.com/commsbio 13