Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells.

Epub ahead of print July 17, 2015 - doi:10.1189/jlb.1A0914-459RR

Article

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells Sae Jin Oh,*,†,‡,1 Sehee Ahn,*,†,1 Young-Hee Jin,†,1 Chieko Ishifune,§ Ji Hyung Kim,*,†,2 Koji Yasutomo,§,3 and Doo Hyun Chung*,†,‡,3 *Department of Pathology, ‡Ischemic/Hypoxia Institute, and †Laboratory of Immune Regulation, Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea; and §Department of Immunology and Parasitology, Institute of Health Biosciences, University of Tokushima Graduate School, Kuramoto, Tokushima, Japan RECEIVED SEPTEMBER 30, 2014; REVISED JUNE 29, 2015; ACCEPTED JUNE 29, 2015. DOI: 10.1189/jlb.1A0914-459RR

ABSTRACT Invariant natural killer T cells are a distinct subset of T cells that exert Janus-like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4-specific gene ablation of Notch 1 and Notch 2 (N1N22/2) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N22/2 mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.12CD44+ to the NK1.1+CD44+ stage, resulting in accumulation of NK1.12 CD44+ invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N22/2 mice produced lower cytokine levels and increased apoptosis versus wild-type invariant natural killer T cells. Furthermore, Notch 1/Notch 2-deficient, but not wild type, invariant natural killer T cells failed to promote antibody-induced arthritis in CD1d2/2 mice. Unlike N1N22/2 mice, RBP-jlox/lox CD4-Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild-type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP-j in the thymus. J. Leukoc. Biol. 98: 000–000; 2015.

Introduction iNKT cells are a distinct subset of T cells characterized by the expression of semi-invariant Va14-Ja18 TCRs and natural killer

Abbreviations: a-GalCer = a-galactosylceramide, BM = bone marrow, i NKT = invariant natural killer T cell, N1N22/2 = CD4-specific knockout of Notch 1 and Notch 2, PLZF = promyelocytic leukemia zinc finger, RBP-j = recombination signal binding for immunoglobulin k J region, Treg = regulatory T cell, WT = wild-type The online version of this paper, found at www.jleukbio.org, includes supplemental information.

0741-5400/15/0098-0001 © Society for Leukocyte Biology

receptors. iNKT cells recognize a-GalCer, a glycolipid derived from a marine sponge, presented by CD1d expressed on antigenpresenting cells in the periphery and cortical CD4+CD8+ doublepositive thymocytes [1, 2]. Upon activation, iNKT cells rapidly secrete large amounts of various cytokines, such as IL-4 and IFN-g in vitro and in vivo [3, 4]. Thus, iNKT cells have been suggested to have a key role in immune regulation by balancing the Th1/ Th2 microenvironment. In the thymus, iNKT cells differentiate from the double-positive stage into CD42CD82 double-negative or CD4+ single positive iNKT cells [5]. Several molecules associated with signal transduction and transcription factors are known to regulate thymic development of iNKT cells [6]. Notch signaling, originally characterized in flies, has an important role in cell-to-cell interactions during development [7]. In mammals, 4 Notch proteins have been reported and they bind 5 ligands of the Jagged (Jagged 1 and Jagged 2) and D-like ligand (DLL1, DLL3, and DLL4) families [7]. Upon engagement by ligands, Notch proteins provide signals via RBP-j (also known as CSL in humans)-mediated canonical and/or noncanonical pathways [8]. Notch molecules are important for T cell lineage commitment and early thymic development, up to the doublenegative 3 stage [7] and also affect T cell effector functions [7, 9–12] as well as the expansion of Tregs [13]. Thus, Notch regulates the development and functions of various subsets of T cells in vivo. Recently, hepatic NK1.1+CD3+ NKT cells were found to express Notch 1 and Notch 2, suggesting that Notch signaling might contribute to the development and functions of iNKT cells [14]. However, it has not yet been determined whether Notch regulates thymic differentiation or the effector functions of iNKT cells in vitro or in vivo. To address this, we investigated the functional roles of Notch 1 and Notch 2 in iNKT cells in vitro and in vivo.

1. These authors contributed equally to this work. 2. Current affiliation: Harvard Medical School, Boston, Massachusetts, USA 3. Correspondence: D.H.C., Department of Pathology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea. E-mail: [email protected]; K.Y., Department of Immunology and Parasitology, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15, Kuramoto, Tokushima 770-8503, Japan. E-mail: [email protected]

Volume 98, November 2015

Journal of Leukocyte Biology 1

Copyright 2015 by The Society for Leukocyte Biology.

MATERIALS AND METHODS

ELISA

Mice

Cytokine levels in cell culture supernatants were measured using a BD Bioscience ELISA kit according to the manufacturer’s protocol.

All mice used in this study were from the C57BL/6 background. C57BL/6 mice (7–8 wk old) were purchased from the Orient Company Ltd. (Seoul, Korea). N2lox/lox mice were obtained from Dr. Shigeru Chiba (University of Tokyo, Tokyo, Japan), as described previously [15]. N1lox/lox, CD4-Cre, RBP-jlox/lox, CD1d2/2, and C57BL/6 (45.2) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). N1N22/2 and RBP-j2/2 mice were generated by backcrossing N1lox/lox and N2lox/lox or RBP-jlox/lox and CD4-Cre mice, respectively. Moreover, deletion of RBP-j in RBP-j2/2 mice was confirmed in thymic iNKT cells using RT-PCR (Supplemental Fig. 1A). Three lines of mice (N1, N2, RBP-j knockout mice) have been backcrossed with B6 mice at least 7 times. The mice were bred and maintained under specific pathogen-free conditions at the Biomedical Research Institute (Seoul National University Hospital, Seoul, Korea). All animal experiments were approved by the Institutional Animal Care and Use Committee at Seoul National University Hospital.

To perform RT-PCR, total RNA was isolated from iNKT cells using an RNeasy kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. iNKT cells were sorted using the MACS bead system. RNA was reverse transcribed into cDNA using Maloney murine leukemia virus reverse transcriptase Taq polymerase (Promega, Madison, WI, USA) before PCR. For quantitative RT-PCR, gene-specific PCR products were measured using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The following primers and probes were synthesized by Applied Biosystems: GAPDH (4352339E), IL-4 (Mm00445259_m1), IFN-g (Mm01168134_m1), TGF-b (Mm01178820_m1), RBP-j (Mm03053645_s1), BCL2 (Mm00477631_m1), BCLXL (Mm01252355_m1), CXCR6 (Mm02620517_s1), and Id2 (Mm00711781_m1). Gene expression levels were normalized to GAPDH.

Cell preparation, antibodies, and flow cytometry

Serum transfer and arthritis scoring

Single-cell suspensions were prepared from the thymus, spleen, and cervical lymph nodes. Mononuclear cells from livers were isolated using Percoll gradient centrifugation. iNKT cells in N1N22/2, RBP-j2/2, and WT mice were detected using APC-conjugated a-GalCer/CD1d tetramers unloaded or loaded with PBS57 obtained from the tetramer facility of the U.S. National Institutes of Health (Bethesda, MD, USA) (Supplemental Fig. 1B). a-GalCer/ CD1d tetramer+CD3+ iNKT cells were sorted from the thymus and livers using FACSAria II (Becton Dickinson, San Jose, CA, USA; purity .99%). Alternatively, liver mononuclear cells were labeled with APC-conjugated a-GalCer/CD1d tetramers, bound to anti-APC magnetic beads, and enriched on a MACS separator (Miltenyi Biotec, Auburn, CA, USA; purity .89%). Various antibodies were used: FITC-conjugated anti-CD80 (16-10A1), antiCD86 (B7-2), anti-CD3 (145-2C11), anti-CD24, PE-conjugated anti-CD1d (1B1), anti-Notch 1 (mN1A) (BD Biosciences, Sparks, MD, USA), anti-Notch 2 (HMN2-35) (BioLegend, San Diego, CA); FITC- or eFluor450-conjugated antiCD4 (GK1.5), anti-CD44 (IM7), PE- or PerCP-cy5.5-conjugated anti-NK1.1 (PK136), anti-CD45.1 (A20), and Alexa Fluor 700-conjugated anti-CD45.2 mAbs (104) (eBioscience, San Diego, CA, USA). To estimate apoptosis, freshly isolated cells from the thymus and livers were stimulated for 3 h with PMA (1 ng/ml; Invitrogen, Carlsbad, CA, USA) and ionomycin (1 mM; Invitrogen), and then stained with APC-conjugated a-GalCer/CD1d tetramers and FITC-conjugated anti-CD3 mAb, washed, and stained with PE-conjugated Annexin V, according to the manufacturer’s protocol. Flow cytometry was performed with an LSRII flow cytometer (BD Biosciences), and data were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).

Recipient mice were injected intraperitoneally with 150 ml pooled K/BxN sera on days 0 and 2. Ankle thickness was measured with calipers (Manostat, Herisau, Switzerland). CD1d2/2 mice were injected intravenously with 5 3 105 iNKT cells enriched by magnetic sorting with PBS57-CD1d tetramers 1 d before administering K/BxN sera. Joint swelling was monitored and scored as reported previously [16, 17].

Generation of mixed BM chimeras BM cells were prepared from the femurs and tibias of WT B6 (CD45.2+) or N1N22/2 B6 (CD45.1+) donor mice and mature T cells were depleted. Recipient mice (CD45.2+) were lethally irradiated (800 rads) and injected intravenously with a 1:1 mixture of BM cells from WT or N1N22/2 mice (2 3 106 cells). Chimeras were analyzed 7 wk after BM transplantation.

Intracellular staining for cytokines and transcription factors Cells were stained with PBS57-CD1d tetramers, anti-CD3, anti-CD4, and antiNK1.1 mAbs, fixed, then permeabilized with fixation and permeabilization buffers (BD Biosciences). The cells were stained using PE-conjugated anti-Rat IgG1k, anti-IL-4, anti-IFN-g, anti-PLZF (Mags.21F7), anti-Gata3 (TWAJ), antiKi67 (SolA15) mAbs, PE- or PE-Cyanine7 conjugated anti-T bet (4B10), and PE- or perCP-eFluor 710 conjugated anti-ROR-gt (B2D) mAbs purchased from eBioscience. To estimate cytokine production, cells were stimulated for 4 h with PMA and ionomycin in the presence of Golgi stop and Golgi plug (BD Pharmingen, San Diego, CA, USA).

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RT-PCR

Statistical analyses Statistical significance was analyzed using the Prism software (version 5.0; GraphPad Software, La Jolla, CA, USA). Unpaired 2-tailed t test or 2-way ANOVA was performed to compare the groups. A P value , 0.05 was considered statistically significant.

RESULTS

Notch 1 and Notch 2 synergistically regulate differentiation of iNKT cells in the thymus To explore whether Notch 1 and Notch 2, separately or in combination, are involved in the development of iNKT cells in vivo, the populations of iNKT cells in the thymus, livers, spleens, and lymph nodes from N1lox/lox CD4-Cre (N12/2), N2lox/lox CD4-Cre (N22/2), and double-mutant N1N2lox/lox CD4-Cre mice (referred to hereafter as N1N22/2) were assessed. It has been reported that CD4-Cre-directed deletion of Notch 1 or presenilin does not alter percentages and absolute numbers of total thymocytes [18, 19]. Consistently, N12/2 mice showed similar thymic development of iNKT cells in percentages and numbers during development (stages st1, NK1.12CD442, st2, NK1.12CD44+, st3, NK1.1+CD44+) compared with WT mice (Fig. 1A). In contrast to N12/2 mice, N22/2 and N1N22/2 mice demonstrated higher percentages and numbers of iNKT cells in the thymus than WT mice did (Fig. 1A and B and Supplemental Fig. 1C). Moreover, percentages of NK1.12CD44+iNKT cells (stage 2) in the thymus were increased in N1N22/2 mice, whereas those of NK1.1+CD44+ iNKT cells (stage 3) were reduced (Fig. 1B). However, numbers of thymic iNKT cells at stages 2, and 3 from N1N22/2 mice were higher than those of WT mice, which may reflect the high numbers of thymic iNKT cells in N1N22/2 mice. These findings indicate that deficiency of both Notch 1 and Notch 2 in iNKT cells perturbs maturation from stage 2 to 3 in the thymus, although Notch 1 alone does not affect the thymic development of iNKT cells.

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Oh et al. Notch signaling regulates maturation of iNKT cells

Figure 1. Development of iNKT cells in the thymus is perturbed in N1N2lox/lox CD4-Cre (N1N22/2) mice. (A) The percentages of iNKT cells from the thymus of WT, N1lox/lox CD4-Cre (N12/2), N2lox/lox CD4-Cre (N22/2), and N1N22/2 mice were measured by flow cytometry (n = 10). (B) Percentages and numbers of total iNKT cells or those in developmental stages (st1, NK1.12CD442; st2, NK1.12CD44+; st3, NK1.1+CD44+) from the thymus of WT or N1N22/2 mice are presented (n = 6). (C) Expression levels of PLZF (n = 10), T bet (n = 8), GATA-3 (n = 8), and ROR-gt (n = 8) in iNKT cells at thymic developmental stages were measured in WT and N1N22/2 mice by flow cytometry. (D) A subset analysis of NKT1, NKT2, and NKT17 in the thymus from WT or N1N22/2 mice. The diagrams of flow cytometry and the graphs present the percentages for each subset in the thymus. The percentages for the subsets were calculated from flow cytometric analysis (n = 8). (A–D) The results shown are representative of 3 independent experiments. n.s., not significant; *P , 0.05; **P , 0.01; ***P , 0.001, unpaired 2-tailed t test.

Several transcription factors have important roles in the development and functions of iNKT cells [6]. Thus, to functionally link Notch signaling and other transcription factors in iNKT cells, we evaluated the expression of several transcription factors in iNKT cells from the thymus. PLZF, T bet, GATA-3, and ROR-gt expression was similar in thymic iNKT cells from N22/2 and WT mice (Supplemental Fig. 1D). In contrast, the expression levels of PLZF in thymic NK1.12CD442 iNKT cells (stage 1) in N1N22/2 mice were higher than they were in WT mice, whereas those in NK1.12CD44+iNKT cells (stage 2) of N1N22/2 mice were lower (Fig. 1C and Supplemental Fig. 2A). However, the levels of T bet, GATA-3, and ROR-gt expression were similar in both groups. Consistent with these findings, thymic iNKT cells from N1N22/2 or RBP-J2/2 mice similarly produced IL-4 or IFN-g to WT iNKT cells upon stimulation (Supplemental Fig. 2B). These findings suggest that ablation of Notch signaling in

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iNKT cells alters the expression levels of PLZF, depending on their developmental stages. IL-17 receptor B-expressing iNKT cells have been reported to be distinct in their development and functions compared with classic iNKT cells [20], suggesting that iNKT cells are functionally heterogeneous in terms of cytokine production. Furthermore, Lee et al. [21] defined distinct subsets of iNKT cells as NKT1, NKT2, and NKT17, which produced IFN-g, IL-4, and IL-17, respectively, in the thymus. Thus, they suggested that these subsets might represent diverse lineages, but not developmental stages [21]. In our experiments, percentages of thymic NKT17 in N1N22/2mice were found to be higher than those in WT mice, whereas there was no difference in NKT1 and NKT2 of the thymus between WT and N1N22/2 mice (Fig. 1D). In lymph nodes, iNKT cells from N1N22/2 mice decreased NKT1, but increased NKT17 compared with WT mice (Supplemental Fig. 3A). Consistently, production of



IL-17 by iNKT cells of N1N22/2 mice was enhanced upon a-GalCer stimulation compared with WT mice (Supplemental Fig. 3B). These findings suggest that Notch 1 and Notch 2 might be involved in generation of NKT 17 in the thymus and lymph nodes.

Notch 1 and Notch 2 synergistically regulate homeostasis and maturation of iNKT cells in the periphery In the liver, percentages of iNKT cells in N12/2 mice were similar to the percentages in WT mice, whereas N22/2 mice demonstrated a reduction in iNKT cell percentages and numbers vs. WT mice (Fig. 2A and Supplemental Fig. 1C). However, the percentages of iNKT cells in the lymph nodes and spleens from N12/2, N22/2, and N1N22/2 mice were not altered compared with WT mice (Fig. 2B, C). Furthermore, the numbers and percentages of total iNKT cells in the livers from N22/2 and N1N22/2 mice were significantly reduced compared with those from WT mice. These findings suggest that Notch 1 and Notch 2 synergistically regulate peripheral homeostasis of iNKT cells,

although Notch 2 alone, but not Notch 1, may be involved in this process. The critical transition from NK1.12 to NK1.1+ usually takes place after immature NK1.12 iNKT cells emigrate to peripheral tissues [22, 23], indicating that NK1.1+CD4+ iNKT cells may represent a subset of mature iNKT cells in the periphery. The percentages of mature NK1.1+CD4+ iNKT cells in the livers, spleen, and lymph nodes from N1N22/2 mice were lower than they were in WT mice, whereas those of immature NK1.12CD4+ iNKT cells were higher (Fig. 2A–C). Moreover, numbers of mature NK1.1+CD4+ iNKT cells in the livers from N1N22/2 mice were lower than they were in WT mice (Fig. 2A), suggesting that Notch 1 and Notch 2 contribute to the maturation of iNKT cells in the periphery. Apoptosis in Notch 1/Notch 2-deficient iNKT cells from the liver, but not the thymus, was increased compared with WT iNKT cells in the presence or absence of stimulation with PMA/ionomycin (Fig. 2D and Supplemental Fig. 4A), although hepatic iNKT cells are prone to die upon removal from the liver [24]. Furthermore, RT-PCR revealed that the expression of antiapoptotic molecules, such as BCL2, BCLXL,

Figure 2. N1N2lox/lox CD4-Cre (N1N22/2) mice show dysregulated homeostasis and increased apoptosis of iNKT in the periphery. (A) The percentages or numbers or both in the iNKT cells from the livers of WT, N1lox/lox CD4-Cre (N12/2), N2lox/lox CD4-Cre (N22/2), and N1N22/2 mice were assessed by flow cytometry (left panels) and are presented as graphs (right panels). The percentages and numbers of iNKT cells from the spleens (B) and lymph nodes (C) of WT or N1N22/2 mice were estimated and are presented as flow cytometry diagrams (left panels) and graphs (right panels). (A–C) The percentages and numbers of CD4+NK1.1+ iNKT cells from these mice were also estimated [n = 6 (A); n = 8 (B and C)]. (D) Apoptosis of hepatic iNKT cells from WT or N1N22/2 mice was evaluated by measuring the expression levels of Annexin V (left panels) and the percentages of Annexin V+ iNKT cells (right panels) in the presence or absence of stimulation with PMA/ionomycin (n = 5). (E) The mRNA levels of BCL2, BCLXL, CXCR6, and Id2 were measured in hepatic iNKT cells from WT or N1N22/2 mice by RT-PCR (n = 5). (F) Expression levels of PLZF, T bet, GATA-3, and ROR-gt in iNKT cells from the livers were measured in WT and N1N22/2 mice by flow cytometry (n = 8). (G) Expression levels of T bet in CD4+ NK1.1 2 and CD4+NK1.1+ iNKT cells were analyzed based on results in panel F. (A–F) The results shown are representative of 3 independent experiments. n.s., not significant; *P , 0.05; **P , 0.01; ***P , 0.001, unpaired 2-tailed t test.

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CXCR6, and Id2 was lower in sorted Notch 1/Notch 2-deficient iNKT cells from the livers than WT iNKT cells in the absence of additional stimulation (Fig. 2E). However, the expression levels of Ki-67 in iNKT cells from the thymus and livers of N1N22/2 mice were similar to those of WT mice, suggesting that Notch 1 and Notch 2 minimally affect proliferation of iNKT cells in the thymus and liver (Supplemental Fig. 4C). Combined, it is likely that hepatic iNKT cells from N1N22/2 mice appear to be more prone to die compared with those of WT iNKT cells. Among the transcription factors tested, T bet expression in total hepatic iNKT cells from N1N22/2 mice was lower than in WT iNKT cells (Fig. 2F). Moreover, CD4+NK1.12 iNKT cells showed lower T bet expression than those from WT mice, whereas CD4+NK1.1+ iNKT cells expressed T bet similarly in both groups (Fig. 2G).

produced less IL-4 and IFN-g than did WT iNKT cells (Fig. 3A). Consistent with cytokine production, intracellular staining revealed that the percentages of iNKT cells producing either IL-4 or IFN-g were significantly lower in N1N22/2 mice than they were in WT mice (Fig. 3B), indicating that Notch 1/Notch 2-deficient iNKT cells have a defect in activation-induced cytokine production in vitro. Furthermore, Notch 1/Notch 2-deficient iNKT cells failed to promote antibody-induced arthritis in CD1d2/2 mice, whereas WT iNKT cells enhanced it to the level of WT mice by increasing IL-4 and IFN-g production and suppressing TGF-b production in the joints (Fig. 3C and D), as reported previously [16, 17]. Combined, these results suggest that Notch 1/Notch 2-deficient iNKT cells have a defect in activation-induced IL-4 and IFN-g production in vitro and in vivo.

Upon activation, Notch 1 and Notch 2 regulate cytokine production by iNKT cells

Intrinsic deficiency of Notch 1 and Notch 2 in iNKT cells perturbs thymic development and maturation in the liver

To investigate whether ablation of both Notch 1 and Notch 2 affects iNKT cell functions, sorted hepatic iNKT cells from WT or N1N22/2 mice were stimulated using anti-CD3 and CD28 mAbs. Upon activation, hepatic iNKT cells from N1N22/2 mice

To confirm that an intrinsic deficiency of Notch 1 and Notch 2 affected thymic development and peripheral maturation of iNKT cells, we generated mixed BM chimera mice using lethally

Figure 3. Notch 1 and Notch 2 regulate cytokine production by iNKT cells in the liver. (A and B) iNKT cells (5 3 105) sorted from the livers of WT or N1N2lox/lox CD4-Cre (N1N22/2) mice were stimulated with coated anti-CD3 (5 mg/ml) and anti-CD28 (5 mg/ml) mAbs (A) or PMA and ionomycin (B). The levels of IL-4 and IFN-g in the culture supernatants and cytosolic expression were assessed using ELISA (A) and intracellular staining was used to compare with isotype-matched IgG controls (B), respectively. Moreover, the percentages of hepatic IL-4+ or IFN-g+ iNKT cells were also estimated in WT or N1N22/2 mice based on flow cytometry analysis [n = 4 (A); n = 6 (B)]. (C and D) To cause antibody-induced arthritis in mice, K/BxN serum was injected into WT, CD1d2/2, or CD1d2/2 mice and adoptively transferred with WT or N1N2 2/2 iNKT cells. iNKT cells were sorted from WT or N1N22/2 mice and adoptively transferred into CD1d2/2 mice 1 d before injection of K/BxN serum. Ankle thickness and clinical index were measured (C), and cytokine expression in the joints was estimated 9 d after K/BxN serum injection using RT-PCR (D) [n = 6 (C); n = 6 (D)]. (A–D) The results shown are representative of 3 independent experiments. n.s., not significant; *P , 0.05; **P , 0.01; ***P , 0.001; unpaired 2-tailed t test (A, B, and D) and 2-way ANOVA (C).

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irradiated WT recipient mice in a 1:1 ratio (CD45.2+ WT/ CD45.1+ N1N22/2 BM cells; Fig. 4A). Consistent with N1N22/2 mice, the percentages of CD45.1+ iNKT cells (N1N22/2) in the thymus of chimeric mice were higher than those in CD45.2+ iNKT cells (WT; Fig. 4B). Among the population of CD45.1+ iNKT cells (N1N22/2), NK1.1+CD44+ iNKT cells in the thymus were reduced, compared with CD45.2+ iNKT cells (WT), whereas NK1.12CD44+ iNKT cells were increased slightly (Fig. 4C). In the livers of chimeric mice, the percentages of CD45.1+ iNKT cells (N1N22/2) were lower than those in CD45.2+ iNKT cells (WT; Fig. 4D). Moreover, the percentages of mature CD4+NK1.1+ iNKT cells among hepatic CD45.1+ iNKT cells (N1N22/2) were

lower than those in CD45.2+ iNKT cells (WT; Fig. 4E). However, percentages of CD45.1+ and CD45.2+ conventional T cells were similar in the thymus and liver of chimeric mice (Fig. 4B and D). Meanwhile, WT and N1N22/2 mice expressed similar levels of CD1d, CD80, and CD86 on a-GalCer/CD1dtet2 conventional CD4+CD8+ thymocytes (Supplemental Fig. 5A). iNKT cells from the thymus and livers demonstrated comparable expression of Notch 1, Notch 2, and RBP-j as conventional CD4+ T cells (Supplemental Fig. 5B and C). Thus, it seems that the defect in iNKT cell maturation in the thymus and livers of N1N22/2 mice is due to a failure in Notch-mediated signaling, rather than TCR stimulation, in iNKT cells. Collectively, these findings indicate

Figure 4. Intrinsic deficiency of Notch 1 and Notch 2 in iNKT cells perturbs thymic development, homeostasis, and maturation in the liver. (A) The diagram shows the experimental scheme to generate BM chimeric mice using WT and N1N2lox/lox CD4-Cre (N1N22/2) mice. Moreover, flow cytometric analysis of the mixture of WT and N1N22/2 BM cells was performed to estimate CD45.1+ vs. CD45.2+ populations before injection. iNKT and conventional T cells obtained from the thymus (B) or liver (D) of BM chimeric mice were analyzed for CD45.1 and CD45.2 expression. The percentages of iNKT cells in these mice are presented. (C) The percentages of iNKT cells at developmental stages in gated CD45.1+ or CD45.2+ iNKT cells from the thymus of chimeric mice were estimated from CD44 and NK1.1 expression. (E) Expression patterns of NK1.1 were evaluated in gated CD45.1+ or CD45.2+ iNKT cells from the liver of chimeric mice. (B–E) n = 10. (C and E) The percentages of iNKT cells were calculated based on flow cytometry analysis and are presented as bar graphs. (A–E) The results shown are representative of 3 independent experiments. *P , 0.05; **P , 0.01; ***P , 0.001, unpaired 2-tailed t test.

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that intrinsic expression of Notch 1 and Notch 2 regulates maturation in the thymus and periphery of iNKT cells.

RBP-jlox/lox CD4-Cre (RBP-j2/2) mice show comparable thymic development to WT mice, but peripheral maturation and functional defects in iNKT cells Upon engagement of Notch on the cell surface, proteolytic cleaved intracellular domain of Notch translocates the nucleus and binds to RBP-j, which plays critical roles in the signal pathway of Notch [25]. Therefore, we investigated the development and functions of iNKT cells in RBP-j2/2 mice. Unlike N1N22/2 mice,

RBP-j2/2 mice showed similar percentages and numbers of total thymic iNKT cells, levels of apoptosis, and transcription factor expression as those of WT mice had. However, RBP-j2/2 mice demonstrated higher percentages and numbers of iNKT cells at stage 2 in the thymus than WT mice did. The percentages, but not the numbers of iNKT cells, at stage 3 were reduced (Fig. 5A) in RBP-j2/2 mice compared with WT mice. These results suggest that thymic development of iNKT cells might be partly independent of RBP-j (Fig. 5A and Supplemental Figs. 4B and 5D). In contrast to the thymus, the percentages and numbers of total and mature NK1.1+CD4+ iNKT cells from the liver were decreased in RBP-j2/2

Figure 5. RBP-jlox/lox CD4-Cre (RBP-j2/2) mice showed similar numbers of thymic iNKT cells as those shown by WT mice, but they had defects in homeostasis, maturation, and function of hepatic iNKT cells. (A and B) The percentages and numbers of iNKT cells from the thymus (A) and liver (B) of WT and RBP-j2/2 mice were measured. These results are presented as flow cytometry diagrams (left panels) and graphs (right panels). Thymic developmental stages of iNKT cells are defined in terms of expression of CD44 and NK1.1: st1, NK1.12CD442; st2, NK1.12CD44+; and st3, NK1.1+CD44+. (C and D) Hepatic iNKT cells sorted from WT or RBP-j2/2 mice were activated with coated anti-CD3 (5 mg/ml) and anti-CD28 (5 mg/ml) mAbs (C) or with PMA and ionomycin (D). The levels of IL-4 and IFN-g in the culture supernatants (C) and intracellular staining for these cytokines (D) were assessed. Moreover, the percentages of hepatic IL-4+ or IFN-g+ iNKT cells were estimated in WT and RBP-j2/2 mice. (A–D) n = 6. (E) The expression levels of PLZF, T bet, GATA-3, and ROR-gt in iNKT cells from the livers of WT and RBP-j2/2 mice were measured by flow cytometry (n = 7). (F) Apoptosis in iNKT cells from the liver of RBP-j2/2 or WT mice was evaluated by measuring the expression levels of Annexin V (left panels) and the percentages of Annexin V+ iNKT cells (right panels) in the presence or absence of stimulation with PMA/ionomycin (n = 5). (G) The mRNA levels of CXCR6 and Id2 were measured in hepatic iNKT cells from WT or RBP-j2/2 mice using RT-PCR (n = 5). The results shown were analyzed using data collected from 3 independent experiments (n = 2 in each experiment) (A) and a representative of 3 independent experiments (B–G). n.s., not significant; *P , 0.05; **P , 0.01; ***P , 0.001, unpaired 2-tailed t test.

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mice compared with WT mice (Fig. 5B), consistent with that of the N1N22/2 mice. Moreover, total hepatic iNKT cells from RBP-j2/2 mice produced less IL-4 and IFN-g (Fig. 5C). The percentages of iNKT cells producing IL-4 or IFN-g were lower in RBP-j2/2 mice than they were in WT mice (Fig. 5D). Thymic iNKT cells from RBP-j2/2 mice expressed PLZF, T bet, GATA-3, and ROR-gt, similar to the expression in WT mice, whereas hepatic RBP-jdeficient iNKT cells showed lower levels of T bet expression (Fig. 5E and Supplemental Fig. 5D). Moreover, RBP-j2/2 mice showed increased apoptosis of hepatic iNKT cells, compared with WT iNKT cells in the presence or absence of activation (Fig. 5F). Consistently, mRNA expression of anti-apoptotic molecules, such as CXCR6, and Id2, was less in sorted RBP-j-deficient iNKT cells from the liver than their expression in WT iNKT cells (Fig. 5G). These findings suggest that the maturation, homeostasis, and functions of iNKT cell in the periphery might depend on RBP-j, in contrast to that found for thymic development.

DISCUSSION In our experiments, N1N22/2 mice showed an accumulation of NK1.12CD44+ (stage 2) iNKT cells in the thymus compared with WT mice, which might be associated with increased numbers of thymic iNKT cells in N1N22/2 mice. Moreover, this accumulation of iNKT cells at stage 2 might be caused by impaired thymic maturation of iNKT cells from the NK1.12CD44+ to the NK1.1+CD44+ stages in N1N22/2 mice. Alternatively, it is also feasible that the expansion of iNKT cells predominantly occurs within the stage 2 NK1.12CD44+ iNKT compartment in the absence of Notch. However, N1N22/2 and WT mice showed similar Ki-67 expression levels on thymic iNKT cells at 3 developmental stages (Supplemental Fig. 4C), suggesting that expansion of iNKT cells at stage 2 might not be extensive in N1N22/2 mice compared with WT mice. Nevertheless, this issue should be clarified further. The expression level of PLZF in thymic NK1.12CD442 iNKT cells was increased in N1N22/2 mice, whereas it was decreased in NK1.12CD44+iNKT cells of N1N22/2 mice. Among transcription factors that are known to regulate thymic development of iNKT cells [6], it has been reported that PLZF has important roles in early thymic development of iNKT cells [26]. Overexpression of PLZF in mice increased the percentage of thymic iNKT cells like it did in N1N22/2 mice, whereas PLZF-deficient mice showed fewer iNKT cells in the thymus [26]. These findings suggest that altered expression levels of PLZF perturb the thymic development of iNKT cells. Thus, we hypothesized that Notch 1- and Notch 2-associated alteration of PLZF expression might be involved in impaired differentiation of thymic iNKT cells. Unlike the thymus, T bet expression in hepatic CD4+NK1.12 iNKT cells was decreased in N1N22/2 mice. Consistent with this, iNKT cells failed to complete terminal maturation in T bet2/2 mice, leading to fewer iNKT cells in the periphery [27, 28]. Furthermore, ectopic expression of T bet in immature iNKT cells promoted their maturation, driving multiple genes [27]. These findings led to the hypothesis that Notch-mediated regulation of other transcription factors, including T bet, may be associated with peripheral maturation of iNKT cells in vivo. In contrast to the thymus, N1N22/2 mice had fewer iNKT cells and increased 8



apoptosis in the liver. Moreover, the expression of BCL2, BCLXL, CXCR6, and Id2 was decreased in hepatic Notch 1/Notch 2deficient iNKT cells. Consistent with these results, Notch protected CD4+ T cells from apoptosis by inducing broad antiapoptotic gene expression, including BCL2, which promoted CD4+ T cell longevity [29]. Thus, increased apoptosis of hepatic iNKT cells in N1N22/2 mice might partially account for the reduced numbers of Notch 1/Notch 2-deficient iNKT cells in the liver. Like iNKT cells, follicular helper T cells also showed dysregulated homeostasis in the peripheral tissues in N1N22/2 mice, but not in N12/2 or N22/2 mice [30]. In contrast to these cells, a defect in Notch 1 signaling alone perturbs the lineage commitment during thymic development and effector function in conventional T cells in the periphery [31], although Notch 1 and Notch 2 have been reported to contribute synergistically to Th1 cell differentiation against Leishmania major infection [32]. In combination, these findings suggest that synergism of Notch 1 and Notch 2 critically regulates the homeostasis and maturation of nonconventional T cells, such as iNKT and follicular helper T cells. Our experiments demonstrated that, unlike N1N22/2 mice, RBP-j2/2 mice showed similar numbers and percentages of iNKT cells in the thymus, whereas RBP-j-deficient iNKT cells had impaired homeostasis, apoptosis, cytokine production, and transcription factor expression in the liver, consistent with N1N22/2 mice. These findings suggest that thymic development of iNKT cells might be partly independent of RBP-j, whereas Notch signal-mediated alteration of homeostasis, apoptosis, and functions of hepatic iNKT cells depend on RBP-j. These findings suggest that there is discrepancy in the RBP-j-dependency of iNKT cell homeostasis between the thymus and the periphery. Our experiments demonstrated that apoptosis of iNKT cells from liver, but not from the thymus, was increased in RBP-j2/2 mice, compared with WT mice, suggesting that RBP-j-dependent apoptosis regulation might be a candidate for the mechanism explaining this phenomenon. However, this discrepancy might also be attributable to a deficiency in the migration from the thymus to the periphery, activation, proliferation, or some combination of these processes in iNKT cells of RBP-j2/2 mice, which should be clarified further. Recently, it has been demonstrated that Notch exerts effects on various biologic events through a noncanonical pathway [8]. During T cell activation, the intracellular domain of Notch 1 interacts directly with NF-kB and regulates IFN-g expression by a complex formation on the IFN-g promoter in an RBP-j-independent manner [33]. RBP-j-independent Notch signals drive the differentiation of CD4+ Th1 following L. major infection [32]. Furthermore, several studies have demonstrated that NF-kB signaling is important for thymic differentiation of the iNKT cells [34, 35]. Among family members in the NF-kB pathway, ablation of NF-kB1 increased the percentage of NK1.12CD44+ iNKT cells but decreased that of NK1.1+CD44+ iNKT cells as in N1N22/2 mice [36]. Thus, it seems reasonable that iNKT cells may also regulate immune responses through the NF-kB-mediated noncanonical Notch signaling pathway in vivo. In conclusion, our data indicate distinct effects of Notch signaling on iNKT cells in the thymus and liver. We demonstrate that Notch signaling exerts vital effects on thymic development of iNKT cells and their maturation, homeostasis, and function in the periphery, which might be at least partly independent of RBP-j in the thymus.

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AUTHORSHIP S.J.O., S.A., Y.H.J., C.I., and J.H.K. performed the experiments; K.Y. and D.H.C. contributed to the study’s conception and the design of the experiments.

ACKNOWLEDGMENTS This work was supported by a grant from the National Research Foundation of Korea funded by the Korean government (Ministry of Education, Science and Technology) (NRF-2010-0017890) and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (Grant HI14C1277). The authors thank the U.S. National Institutes of Health National Institute of Allergy and Infectious Diseases Tetramer Facility for providing PBS-57-loaded CD1d tetramers. They also thank Taewhan Kim and Jae Sung Ko for technical assistance. DISCLOSURES

The authors declare no competing financial interests.

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