Invariant NKT17 Subsets Differentiation of Invariant NKT2 and Invariant NKT Development Promotes Increased Level of E Protein Ac

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Increased Level of E Protein Activity during Invariant NKT Development Promotes Differentiation of Invariant NKT2 and Invariant NKT17 Subsets This information is current as of September 28, 2021. Taishan Hu, Hongcheng Wang, Amie Simmons, Sandra Bajaña, Ying Zhao, Susan Kovats, Xiao-Hong Sun and Jose Alberola-Ila J Immunol 2013; 191:5065-5073; Prepublished online 11 October 2013; Downloaded from doi: 10.4049/jimmunol.1301546 http://www.jimmunol.org/content/191/10/5065 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/10/11/jimmunol.130154 Material 6.DC1 References This article cites 53 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/191/10/5065.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Increased Level of E Protein Activity during Invariant NKT Development Promotes Differentiation of Invariant NKT2 and Invariant NKT17 Subsets

Taishan Hu,* Hongcheng Wang,* Amie Simmons,* Sandra Bajan˜a,† Ying Zhao,* Susan Kovats,† Xiao-Hong Sun,* and Jose Alberola-Ila*

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates

(GATA-3, RORgT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that Downloaded from regulates the number of iNKT precursors that choose each fate. The Journal of Immunology, 2013, 191: 5065–5073.

nvariant NKT (iNKT) cells are a subset of ab T cells char- transcription factor T-bet, they are also called iNKT1 cells. A acterized by the expression of an invariant Va14-Ja18 TCR reason for this change in nomenclature is the recent realization (Va24-Ja18 in humans) in combination with certain TCRb- that stage 2 cells can choose alternative fates. Besides iNKT1 cells, I + + chains. iNKT cells develop in the thymus from the same pre- they can become RORgT iNKT17 cells (10, 11), or IL-17Rb http://www.jimmunol.org/ cursors as conventional CD4+ and CD8+ ab T cells, CD4+CD8+ iNKT2 cells (12, 13). Thus, after stage 2, iNKT cells mature into double-positive (DP) cells (1), but are selected by lipid Ags pre- differentiated, polarized iNKT1-, iNKT2-, or iNKT17-type cells (6). sented by the nonpolymorphic MHC class I–like molecule CD1d, These cell types have similar cytokine profiles to their Th1/Th2/Th17 present on the surface of other DP thymocytes (reviewed in Refs. counterparts, although their bias is less strict, because iNKT1 are able 2–5). The differentiation of iNKT cells in the thymus has been to produce IL-4. The mechanisms that control this fate choice are not divided into a series of stages, based on phenotypic markers and understood, nor it is known whether differentiation into these line- sensitivity to different mutations (reviewed in Refs. 2, 5–7). After ages is terminal or can be reversed. positive selection, iNKT precursors become CD24hiCD4+CD8lo E proteins are class I basic helix–loop–helix (bHLH) transcrip- CD69hi (“stage 0”). Then they downregulate CD24 and CD8 to tion factors that bind DNA at E-box motifs. Members of the E by guest on September 28, 2021 reach a CD4+CD24loCD44lo stage (“stage 1”). Stage 1 iNKT cells protein family include HEB, E2-2, and two differentially spliced undergo a proliferative burst accompanied by the induction of CD44 products of the E2A gene, E12 and E47. E protein activity is (“stage 2”). At this stage, some immature iNKT cells remain in the negatively regulated by Id proteins. Four members, Id1–Id4, have thymus and mature there, whereas others migrate to the periphery to been identified in mammals, but only Id2 and Id3 are highly complete their differentiation (8, 9). expressed in lymphoid cells (14). E proteins and their Id antago- The majority of the cells that end their differentiation in the nists play important roles in B and T lymphocyte development thymus in the most commonly studied mouse strain, C57BL/6, (reviewed in Refs. 15–17). E and Id proteins also play roles in become CD44+NK1.1+. These cells were called stage 3 iNKTs, iNKT cell development. Id2 deficiency impairs hepatic iNKT cell but given their potential for IFN-g secretion and expression of the survival (18). Specific deletion of HEB, but not E2A, in DP thymocytes impairs iNKT cell development through regulation of DP survival and TCRa recombination (19). *Immunobiology and Cancer Research Program, Oklahoma Medical Research Foun- dation, Oklahoma City, OK 73104; and †Arthritis and Clinical Immunology Program, The Ras/MAPK cascade downstream of the TCR induces Id Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 proteins and inhibits E protein activity to promote thymocyte Received for publication June 12, 2013. Accepted for publication September 13, positive selection (20). Because Ras/MAPK is also required for 2013. iNKT cell development (21), we decided to investigate the effect This work was supported by National Institutes of Health/National Institute of Allergy of maintaining E protein function during iNKT cell development and Infectious Diseases Grant AI059302 (to J.A.-I.), National Institutes of Health using a conditional knock-in mouse model expressing ET2, a fu- Grant AI56129 (to X.-H.S.), and American Heart Association Grant 12SDG8490001 (to T.H.). sion protein between the transactivation domains of E47 and the Address correspondence and reprint requests to Dr. Jose Alberola-Ila and Dr. Xiao- bHLH domain of SCL/Tal1 (22, 23). The bHLH domain of SCL/ Hong Sun, Immunobiology and Cancer Research Program, Oklahoma Medical Tal1 does not mediate homodimerization but has high affinity for Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104. E-mail E proteins, so ET2 competes with Id and SCL/Tal1 proteins to addresses: [email protected] (J.A.-I.) and [email protected] (X.-H.S.) bind to endogenous E proteins. Although ET2 does not exhibit The online version of this article contains supplemental material. transcriptional activity by itself, it acts as a transcriptional acti- Abbreviations used in this article: bHLH, basic helix–loop–helix; DP, double-posi- vator when associated with wild-type E47, so overexpression of tive; iNKT, invariant NKT; MIY, MSCV-IRES-YFP; NLC, normal littermate control; ET2 blocks Id proteins and sustains E protein activity. Nrp-1, Neuropilin-1; S1P1, Sphingosine 1–phosphate receptor 1; Va14 Tg, Va14- This approach has several advantages: it blocks all Id proteins, Ja18 transgenic mice. and recent gene expression profiling shows that in early stages of Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 iNKT cell development, both Id2 and Id3 become induced (24). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301546 5066 E PROTEIN ACTIVITY INFLUENCES iNKT SUBSET CHOICE

Furthermore, conventional Id3 knockout mice have defects in de- of 50 ng/ml phorbol dibutyrate, 500 ng/ml ionomycin, and 3 mMmonensin. velopment of other T cell lineages that indirectly impact iNKT cell Cells were extracellularly stained with anti-TCRb and CD1d tetramer. After development. Global loss of Id3 drives T cell progenitors into the gd washing, cells were intracellularly stained for IL-4 and IFN-g using BD Cytofix/Cytoperm kit (BD Biosciences). T cell lineage, promotes Th17 development, and inhibits naive and regulatory T cell fate (25–29). Finally, as opposed to full knockouts Cytokine detection using FlowCytomix of Id proteins, the effect is partial. E protein activity in positively iNKT cells were enriched as described earlier and plated in anti-CD3– selected ET2-expressing cells is maintained at higher levels than coated plates at 1 3 106/ml in R10 medium (RPMI 1640 with 10% FCS, in cells from normal littermate controls (NLCs), but at lower levels HEPES, penicillin and streptomycin, pyruvate, nonessential amino acids, than in DP thymocytes. L-glutamine, and 2-ME) in the presence of 1 mg/ml anti-CD28 mAb. Using this model, we show that modulation of E protein function Supernatants were collected after 24 or 48 h, and production of IL-4 and IFN-g determined using FlowCytomix kits (eBioscience), according to the plays a role in distinct aspects of iNKT cell development, including manufacturer’s instructions. thymic exit. More interestingly, sustaining E protein activity during positive selection results in an increase in the number of iNKTs that In vitro proliferation assay differentiate into the iNKT2 or iNKT17 subsets at the expense of iNKT cell proliferation was analyzed with the CellTrace Violet Cell Pro- the iNKT1 subset. liferation Kit (Invitrogen). Thymocytes or splenocytes were loaded with 10 mM CellTrace Violet and stimulated with plate-bound anti-CD3 (1 mg/ml) Materials and Methods and CD28 (1 mg/ml). Cells were stained with allophycocyanin-conjugated CD1d-tetramer and PerCp-Cy5.5–conjugated TCRb. Cell proliferation Mice was determined by flow cytometry in viable cells at 4 d stimulation. Data Downloaded from All mice were maintained in a specific pathogen-free facility at Oklahoma were analyzed with FlowJo (Tree Star). Medical Research Foundation and were handled in compliance with guidelines established by the Institutional Animal Care and Use Com- Spleen iNKT viability assay mittees. Generation of ROSA26-ET2 conditional knock-in was described Splenocytes were surface stained with allophycocyanin-conjugated CD1d- previously (30). Expression of ET2 was accomplished through crossing tetramer and PerCp-Cy5.5–conjugated TCRb and then assayed for viability to CD4-Cre Tg mice. Va14-Ja18 transgenic mice (31) (Va14 Tg) were using the Annexin V-PE apoptosis detection kit (eBioscience) according kindly provided by Dr. Albert Bendelac (University of Chicago). CD4- to the manufacturer’s instructions. Zbtb17 transgenic mice have been described previously (32), and were http://www.jimmunol.org/ purchased from The Jackson Laboratory. Mice were used between 8 and 12 Whole-cell extract preparation and EMSA wk of age. Total thymus from NLC and ET-2 mice was stained with CD4 and CD8 Abs, Cell preparation and flow cytometry and CD4+CD8+ small thymocytes (DP) were sorted in a FACSAria. The cell pellet was thawed, resuspended at a concentration of 10 ml/107 cells in Single-cell suspensions were prepared from the thymus, spleen, liver, and cold buffer C, which contained Hepes (20 mM at pH 7.9), NaCl (0.4 M), inguinal lymph nodes. Liver suspensions were purified using a Percoll EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF + protease inhibitors (Amersham Biosciences) gradient. Hepatic mononuclear cells were col- (1 mM), and 1% Nonidet P-40, and vortexed vigorously for 2 min at 4˚C. lected from the 70–40% interface, red cell lysed, and then stained for FACS Debris was pelleted and the supernatant removed as the whole-cell extract. analysis. Cells were incubated with Fc blocking Abs before staining with dsDNA probes were end-labeled using T4 polynucleotide kinase and were specific Abs and tetramer. Dead cells and doublets were excluded from gel purified. Whole-cell extract was used in a gel-shift assay as described by guest on September 28, 2021 analysis. Samples were collected on an LSRII (BD) and analyzed with previously (35). The sequence of the mE5 and Oct-1 oligoprobes was as FlowJo (Tree Star). Intracellular staining to detect expression of SAP, described previously (35). T-bet, Runx-1, Runx-3, Gata-3, PLZF, and RORgT was performed with eBioscience Foxp3 staining kit according to the manufacturer’s instruc- Statistical methods tions. Fluorochrome-labeled mAbs (clone indicated in parentheses) against CD45.1 (A20), CD45.2 (104), TCRb (H57-597), Vb2 (B20.6), Vb7 (TR310), Normal distribution of the data was assessed using the Kolmogorov– Vb8.1,8.2 (MR5-2), CD24 (M1/69), CD1d (1B1), CD4 (L3T4) (GK1.5), Smirnov test. Statistical significance was determined by the Student t test CD8a (ly-2) (53-6.7), CD44 (IM7) NK1.1(PK136), CD19 (bio1D3), SLAMF1 (for parametric data) using GraphPad Prism 6.0. (TC15-12F12.2), SLAMF6 (13G3-19D), IL-4 (11B11) IFN-g (XMG1.2), T-bet (eBio4B10), Gata3 (TWAJ), Runx-1 52(RXDMC), Runx-3 (R3-5G4), RORgT (Q31-378), and PLZF (Mags 21F7) were from eBioscience, Bio- Results legend, or Becton Dickinson. The IL-17Rb Ab (clone 752101) was from R&D iNKT cell development is severely altered in ROSA26ET-2; Systems. CD4-cre knock-in mice iNKT cells were identified using a murine allophycocyanin-, PE- or BV421-conjugated CD1d tetramer loaded with PBS57, an analog of The coding sequence of ET2 was knocked into the ubiquitous a-galactosylceramide (33), provided by the National Institutes of Health ROSA26 locus, preceded by a floxed STOP sequence, and followed Tetramer Facility. All analysis was performed using FlowJo (Tree Star). by an IRES-EGFP element (ROSA26ET-2; Fig. 1A). This mouse Retroviral transduction model has been used to reveal the contrasting role of E and Id proteins in myeloid-versus-lymphoid lineage decisions (30). To drive T-bet and Runx-3 gene were RT-PCR amplified from splenic total RNA and cloned into retroviral MSCV-IRES-YFP (MIY) vector. Preparation of ET2 expression from DP thymocyte stage, we bred ROSA26ET-2 viral supernatant was performed essentially as described previously (34). mice with CD4-cre transgenic mice to create ROSA26ET-2;CD4-cre iNKT cells were enriched by negative selection from total thymocytes of mice. In these animals, ET2 is expressed in DP thymocytes at high Va14 Tg and ET-2;CD4Cre;Va14 Tg mice. Total thymocytes were in- levels, and significantly blocks the inhibitory effect of CD3 stimu- cubated with biotin-conjugated anti-CD8, and DP and CD8+ thymocytes were depleted by negative selection with BD IMagTMStrepavidin Particles lation on E-box DNA binding activity, as shown by EMSA (Fig. 1B). Plus. Magnetic separation was performed according to manufacturer’s ROSA26ET-2;CD4-cre mice (hereafter referred to as “ET-2”) instructions (BD Biosciences). Cells were cultured overnight in RPMI had normal numbers of iNKT cells in the thymus compared with 1640 with 10% FCS and 12.5 ng/ml IL-7 before retrovirus infection. Next NLCs. However, iNKT cells in spleen and liver were dramatically day, cells were infected, incubated at 37˚C for 24 h, and then analyzed by decreased in both percentage and numbers (Fig. 1C). Despite their flow cytometry. normal numbers, thymic iNKT cells in ET-2 mice accumulated Intracellular staining for cytokine detection as CD442NK1.12 and CD44+ NK1.12 cells, traditionally consid- Thymocytes and splenocytes were incubated for 4 h at 4 3 106/ml in R10 ered immature stages (stage 1 and 2) (5, 7) (Fig. 1D). This defect medium (RPMI 1640 with 10% FCS, HEPES, penicillin and streptomycin, is not related to alterations in the expression of molecules in the pyruvate, nonessential amino acids, L-glutamine, and 2b-ME) in the presence SLAMF/SAP pathway or to changes in CD1d expression, and The Journal of Immunology 5067 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Expression of ET2 results in changes in iNKT cell populations. (A) Strategy used to express ET2 in DP thymocytes. (B) E2-box (mE5)– and octamer-binding activity (Oct-1) analyzed by EMSA in whole-cell extracts from preselection DP thymocytes (CD4+CD8+ small) sorted from NLC and ET-2 mice, and stimulated with the indicated Abs for 18 h. (C) Thymocytes, splenocytes, and liver mononuclear cells from NLCs and ET-2-cd4Cre (ET-2) mice were stained with CD4, CD8, PBS57-loaded CD1d tetramer, TCRb, CD44, and NK1.1. The left panel shows the CD1tet-TCRb profiles in thymus (T), spleen (S), liver (L), and inguinal lymph nodes (LN). The right panel shows percentages and total numbers of iNKT cells. (D) The left panel shows the CD44 NK1.1 profile of the thymic iNKT cells gated as in panel (C), and the right panel shows percentages and total numbers of stage 1 (CD442NK1.12), stage 2 (CD44+NK1.12), and stage 3 (CD44+NK1.1+) iNKT cells. Results from nine independent ET-2 and NLC pairs analyzed in five experiments, except for the lymph node, where n = 6. The bar graphs show the average and SEM of all the experiments. Significance as assessed using a two-tailed unpaired t test, ***p , 0.001, **p , 0.01. does not correlate with gross alterations in the Vb repertoire of the thymus at the expense of the more mature stage 3 cells (CD44+ iNKT cells in either spleen or thymus, although there is a small NK1.1+). increase in the percentage of Vb2-bearing iNKTs in ET-2 mice (Supplemental Fig. 1). ET2 iNKT cells have altered cytokine secretion patterns Therefore, maintenance of E protein function during positive iNKT cells at different stages are functionally distinct. Consider- selection of iNKT cells results in the accumulation of stage 1 ing that ET2 iNKT cells have a dramatic developmental defect, (CD442 NK1.12) and stage 2 (CD44+ NK1.12) iNKTs in the we assessed their functional capacity. We tested their response to 5068 E PROTEIN ACTIVITY INFLUENCES iNKT SUBSET CHOICE phorbol dibutyrate and ionomycin stimulation in vitro. As shown in Other possible contributors to the decrease in iNKT cell numbers Fig. 2A, ET-2 iNKT cells in the thymus secrete normal amounts of in the periphery are defects in homeostatic proliferation or sur- IL-4 but little IFN-g after stimulation, whereas ET-2 iNKT cells in vival. Peripheral iNKT cells undergo a slow, IL-15–dependent pro- spleen are defective in both IL-4 and IFN-g secretion. We also liferation (40), and it has been shown that, in the liver, iNKT cell assessed the response of enriched iNKTs from WT and ET-2 mice survival is decreased in Id22/2 mice (18). However, we could to stimulation with plate-bound anti-CD3 + anti-CD28 mAbs, and not detect any increase in apoptosis in splenic iNKT cells in ET-2 observed a similar bias toward IL-4 production in iNKTs from ET- mice (Fig. 3B). These results suggest that the defect in peripheral 2 mice (Fig. 2B). iNKT cell populations is most likely due to a defect in thymic exit We also analyzed the proliferative responses of thymic and resulting from defects in the induction of Nrp-1. splenic NKT in ET-2 and NLC mice (Fig. 2C). Both splenic and ET-2 iNKT cells fail to upregulate IL-2Rb, T-bet, and Runx3 thymic NKTs from ET-2 mice proliferate in response to CD3 + + and CD28 stimulation, although the thymic population consis- Because development of stage 3 iNKT cells (CD44 NK1.1 )is tently proliferated less than iNKTs from the NLC mice. severely blocked in ET-2 mice (Fig. 1C), and IL-15 and IL-2Rb (CD122) expression are required for this transition (40), we ana- The decreased number of ET-2 iNKT cells in the periphery is lyzed CD122 expression in ET-2 iNKT cells. As shown in Fig. 4, due to defects in thymic exit most of the ET-2 iNKT cells in the thymus show a dramatic de- Given the normal numbers of iNKT cells in the thymus, and the fact crease in CD122 expression, compared with normal iNKT cells. that development was apparently arrested at stage 2, when im- During normal development, CD122 is upregulated during stage 2 mature iNKT cells normally exit the thymus to finish their dif- (40) and maximally expressed at stage 3. ET-2 iNKT cells fail to Downloaded from ferentiation, the dramatic decrease of iNKT cell populations in upregulate CD122 at stage 2. However, the few cells that manage spleen and liver was surprising. One possible explanation was a to progress to stage 3 express normal levels of CD122, empha- defect in exit from the thymus. Although the mechanisms that sizing the strong selective pressure at this checkpoint. control thymic exit of stage 2 iNKT cells are not completely de- The observed defects in CD122 expression and IFN-g produc- fined, deletion of Sphingosine 1–phosphate receptor 1 (S1P1) in tion in ET-2 iNKT cells prompted us to analyze the expression of

DP thymocytes results in a blockade in iNKT cell thymic exit T-bet, which regulates these functions in developing iNKT cells http://www.jimmunol.org/ (36), and it has been recently shown that recent thymic emigrant (41), as well as several other transcription factors that may con- iNKT cells express high levels of Neuropilin-1 (Nrp-1) (37), a tribute to this regulation, including Runx-3 (42) and PLZF (32, semaphorin ligand that has been implicated in thymic migration 43). As shown in Fig. 4, total iNKT cells from ET-2 mice have (38, 39). Therefore, we checked for expression of these molecules signiﬁcantly lower levels of T-bet and Runx-3 than WT iNKTs. At in stage 2 thymocytes from NLC and ET-2 mice. As shown in Fig. stage 1, levels are similar, but starting at stage 2, iNKT cells from 3A, although ET-2 stage 2 cells express normal levels of S1P1, ET-2 mice fail to upregulate T-bet and Runx-3. By stage 3, the few they are clearly defective for Nrp-1 expression, suggesting that iNKT cells present in ET-2 mice have mostly normalized the a defect in their migratory ability could explain the quantitative expression of Runx-3 and T-bet. In contrast, whereas PLZF ex- defects in peripheral iNKT cells. pression is lower at stage 1 in ET-2 iNKTs than in NLCs, it does by guest on September 28, 2021

FIGURE 2. iNKT cells from ET-2 mice have altered functional abilities. (A) Thymocytes and splenocytes from NLC and ET-2 mice were incubated for 4 h in the presence of 50 ng/ml phorbol dibutyrate, 500 ng/ml ionomycin, and 3 mM monensin. Cells were extracellularly stained with anti-TCRb and CD1d tetramer. After washing, cells were intracellularly stained for IL-4 and IFN-g. A representative experiment out of four is shown. (B) Enriched iNKT cells from WT or ET-2 mice were cultured in anti-CD3–coated plates in the presence of 1 mg/ml anti-CD28. Forty-eight hours later, the supernatants were collected. IL-4 and IFN-g were measured using FlowCytomix. Bar graphs show the average and SEM of all the experiments (n = 4). Signiﬁcance was assessed using a two-tailed unpaired t test. (C) Thymocytes and splenocytes from NLC and ET-2 mice were loaded with 10 mM CellTrace Violet and stimulated with plate-bound anti-CD3 (1 mg/ml) and -CD28 (1 mg/ml). After 4 d in culture, cells were stained by allophycocyanin-conjugated CD1d- tetramer and PerCp-Cy5.5–conjugated TCRb, and cell proliferation was determined by ﬂow cytometry. Shown is a representative experiment out of three. The Journal of Immunology 5069

FIGURE 3. Altered expression of Nrp-1 and normal survival in ET-2 iNKTs. (A) Two-color density plot showing the expression of Nrp-1 and S1p1 in electronically gated thymic stage 2 iNKTs (TcRb+Tet+ CD44+NK1.12). Shown is one representative experiment out of three (n =5).(B) Percentage of live (Annexin V2), Annexin V+, and Annexin V+ 7-AAD+ splenic iNKT cells in NLC (white bars) and ET-2 (black bars) after 24 h. Bar graphs show the average and SEM of all the experiments (n = 5). Signiﬁcance was assessed using a two-tailed unpaired t test.

not become downregulated in ET-2 stage 2 iNKTs, and even the Therefore, expression of ET2 results in dramatic changes in few ET-2 stage 3 iNKTs have higher levels of PLZF than stage 3 many transcription factors important for NKT cell differentiation. iNKTs from NLC mice. We also analyzed the expression of Runx- These changes occur mostly at stage 2, and include a lack of 1, which has been shown to be required for iNKT cell devel- upregulation of T-bet and CD122, which have been shown to be Downloaded from opment through an unknown mechanism (44). In contrast with required for progress to stage 3 (41), and an increase in the ex- Runx-3, total ET-2 NKTs have increased levels of Runx1, and this pression levels of PLZF, Runx-1, and GATA-3. is due to a lack of downregulation at stage 2 of differentiation. A Many of the CD44+NK1.12 iNKT cells in ET-2 mice are not similar profile was observed for GATA-3, another transcription immature stage 2 iNKT, but iNKT2 or iNKT17 cells factor that has been implicated in NKT cell function (45). + 2 Although stage 2 (CD44 NK1.1 ) iNKT cells in the thymus have http://www.jimmunol.org/ been traditionally considered immature iNKT cells that either exit to the periphery to mature or end their differentiation in the thymus as stage 3 (CD44+NK1.1+) iNKTs (2, 5, 7), recent findings suggest that some of them are actually mature iNKT cells, with a different developmental program (12, 13). These distinct iNKT subsets express IL-17Rb and have either a Th2-like (iNKT2) or a Th17-like (iNKT17) cytokine profile. Given the accumulation of stage 2 cells in the thymus of ET-2 mice, we tested whether they represented cells that had differentiated into these alternative sub- by guest on September 28, 2021 sets, rather than just being blocked at an immature stage. iNKT2 are characterized by expression of IL-17Rb, and it has been suggested that they express high levels of GATA-3 (6), although their initial molecular characterization showed that they expressed comparable levels of GATA-3 to the IL-17Rb2 populations in the thymus (13). Compared with NLCs, thymic iNKT cells from ET-2 mice express higher levels of GATA-3 and IL-17Rb, especially at stage 2 (Fig. 4). Furthermore, as shown in Fig. 5A and 5B, we could detect an increased population of NK1.12 IL- 17Rb+ cells among iNKT cells in the thymus and some peripheral tissues (liver, spleen) from ET-2 mice. Surprisingly, the lymph nodes of ET-2 mice did not contain increased percentages of IL-17Rb+ cells. Analysis of GATA-3 expression in these populations showed that the IL-17Rb+ cells express higher levels of GATA-3 than NK1.1+ IL-17Rb2 or NK1.12 IL-17Rb2 cells (Fig. 5C). How- ever, within these distinct subpopulations, GATA-3 levels are comparable in NLC or ET-2 mice (Fig. 5D). Thus, the high levels of GATA-3 in stage 2 cells in ET-2 mice are due to an accumulation of IL-17Rb+ NKT2s. Another recently described iNKT subset, NKT17 cells are characterized by expression of the transcription factor RORgT (46, 47). Because its precursors are also thought to be included within the FIGURE 4. Altered expression of CD122, IL-17Rb, T-bet, Runx-3, IL-17Rb+NK1.12 population (6), we analyzed the expression GATA-3, and RORgT in ET-2 iNKT cells. Histograms show the expression of RORgT in the NKT populations of NLC and ET-2 mice. As of different genes (named on the top of each column) on electronically gated + total thymic iNKTs (TcRb+; CD1d-tet+) or stage 1 (TcRb+; CD1d-tet+; shown in Fig. 6A and 6C, there are very few RORgT cells within CD442;NK1.12), stage 2 (TcRb+; CD1d-tet+;CD44+;NK1.12), and stage 3 the iNKT populations in the NLC thymus. In contrast, almost half + (TcRb+;CD1d-tet+;CD44+;NK1.1+) iNKT populations (named on the of the iNKT cells present in ET-2 thymi are RORgT , and most of int right). NLC is shown as a solid line; ET-2 is shown as a dashed line. them are NK1.1 . However, within these distinct subpopulations, Shown is one representative set of profiles of five experiments. RORgT levels are comparable between NLC and ET-2 mice 5070 E PROTEIN ACTIVITY INFLUENCES iNKT SUBSET CHOICE

FIGURE 5. A big subset of ET-2 iNKT cells ex- presses IL-17Rb. Flow cytometric analysis of IL- 17Rb expression by NLC and ET-2 iNKT cells. (A) Two-color density plot showing expression of IL- 17Rb and NK1.1 in electronically gated iNKT (TcRb+; CD1d-tet+) cells from thymus (T), liver (L), spleen (S), and inguinal lymph nodes (LN) from NLC and ET-2 mice. (B) Quantification of the pop- Downloaded from ulations shown in (A). Bar graphs show the average and SEM of all the experiments (n = 6). Significance was assessed using a two-tailed unpaired t test. **p , 0.01, ***p , 0.001. (C) Histogram shows the expression levels of GATA-3 in the different iNKT subsets defined by NK1.1 and IL-17Rb.(D) Bar graph shows quantification of GATA-3 expression in the different http://www.jimmunol.org/ iNKT subpopulations in NLC (black bars) or ET-2 (white bars). n = 5. Significance was assessed using a two-tailed unpaired t test. *p , 0.05. by guest on September 28, 2021

(Fig. 6D). Although it has been suggested that RORgT+ NKTs iNKTs, the choice between different iNKT subset differentiation are all IL-17Rb+ (6), we could detect a prominent subset of programs is independent of PLZF levels. RORgT+IL-17Rb2 NKTs in both NLC and ET-2 mice (Fig. One important aspect to consider in the differentiation of lym- 6B). phoid cells in different lineages is the stability of these lineages. These results indicate that the iNKT cells present in the thymus of In the case of ab CD4 Th cells, Th1 and Th2 cells were thought to ET-2 mice are not an accumulation of immature stage 2 iNKT cells, be terminally differentiated. However, it has become apparent in but iNKT2 or iNKT17 cells. Therefore, partial maintenance of E pro- the last few years that the classical Th1 and Th2 lineages are tein DNA binding activity during positive selection of iNKT cells relatively plastic and, under the right conditions, a Th2 cell can be biases their cell fate determination, potentiating iNKT2 or iNKT17 “taught” to secrete IFN-g (48). In principle, iNKTs are thought to cell fate at the expense of the iNKT1 fate. be more plastic because, at least in vitro, a signiﬁcant percentage of them can simultaneously produce IL-4 and IFN-g. The gener- Enforced expression of T-bet and Runx-3 restore IFN-g ation of a sizable population of iNKT2 and iNKT17 cells in ET-2 production in ET-2 iNKT cells provides an opportunity to directly test whether the defect in IFN-g Given the importance of PLZF for iNKT cell development (32, 43), production is only due to the lack of T-bet expression, or if there are and the small defect in its expression observed in stage 1 ET-2 other changes that would make this defect more permanent. iNKT cells, we tested whether forcing its expression would change For these experiments, we introduced T-bet or Runx-3 in im- the subset decision in iNKT ET-2 cells. For these experiments, we mature iNKT cells using retroviruses. To obtain enough cells for bred ET-2 mice to transgenic mice that express PLZF under the these experiments, we bred ET-2 mice with transgenic mice ex- control of the CD4 promoter (CD4-Zbtb16) (32). Expression of pressing a rearranged Va14Ja18 TCRa-chain under the control PLZF at high levels did not rescue the development of iNKT1 of the CD4 promoter (31). Compared with wild-type mice, Va14- cells in ET-2 mice (Supplemental Fig. 2), suggesting that although Ja18+ mice have higher percentages of tetramer-reactive cells in PLZF expression is critical for the developmental program of the thymus, and these iNKT cells have a higher percentage of The Journal of Immunology 5071

resulted in a blockade at stage 2 of differentiation, similar to that observed in a wild-type TCR repertoire (see Supplemental Fig. 3). Enriched iNKT cells from Va14Ja18 and Va14Ja18;ET-2 mice were infected in vitro with T-bet or Runx-3 retroviruses and cultured. After 1 d, we assessed the expression of CD122 and NK1.1, as well as their cytokine production after stimulation. As shown in Fig. 7, overexpression of T-bet significantly upregulates CD122 expression and restores IFN-g production to Va14Ja18;ET-2 cells. Runx-3 increases CD122 expression, although the magni- tude of the effect is smaller and fails to achieve statistical significance, and also restores IFN-g production in Va14Ja18;ET-2 iNKTs. In contrast, NK1.1 expression could not be rescued by either transcription factor. These experiments suggest that the NKT1, NKT2, NKT17 subset choice has the potential to be relatively plastic, and stimuli that turn on T-bet could change the cytokine profile of a differentiated NKT2 cell, as has been shown for Th2 cells (48).

FIGURE 6. ET-2 iNKT cells express RORgT. Flow cytometric analysis of Downloaded from RORgT expression by NLC and ET-2 iNKT cells. (A) Two-color histogram Discussion showing expression of RORgTandNK1.1inelectronicallygatediNKT Although a lot is known about the requirements for iNKT cell de- + + B (TcRb ; CD1d-tet ) cells from NLC and ET-2 mice. ( ) Two-color histogram velopment, the molecular mechanisms that control development of showing expression of RORgT and IL-17Rb in electronically gated iNKT cells the recently described different subsets of iNKTs (iNKT1, iNKT2, from NLC and ET-2 mice. (C) Quantification of the populations shown in (A) and (B). Bar graphs show the average and SEM (n = 6). Significance was iNKT17) are not understood (6). In this work, we provide evidence that alterations in the levels of E protein activity during the early assessed using a two-tailed unpaired t test. **p , 0.01, ***p , 0.001, ****p , http://www.jimmunol.org/ 0.0001. (D) Bar graphs show the mean fluorescence intensity for RORgT stages of iNKT cell differentiation bias this process. Higher levels of in different thymic subpopulations in NLC or ET-2 mice, including NKT E protein activity sustained during iNKT cell development promote RORgThi,NKTRORgTlo, DP thymocytes, and TCRhi single-positive thymo- the IL-17Rb+,GATA-3hi iNKT2, and RORgT+ iNKT17 lineages, at cytes. (n = 5). Significance was assessed using a two-tailed unpaired t test. the expense of the IL2Rb+,T-bethi iNKT1 lineage. To manipulate E protein activity, we expressed a chimeric tran- HSAhi and NK1.12 populations, probably because many more scription factor, ET2, composed of the transactivation domains iNKT cells are undergoing positive selection. Expression of ET2 of E47 and the bHLH domain of SCL/Tal (22, 23) (Fig. 1A). The by guest on September 28, 2021

FIGURE 7. Forced expression of T-bet or Runx-3 in ET-2 iNKT cells restores IFN-g production. Enriched iNKT cells were infected in vitro with retrovirus encoding for T-bet or Runx-3 and after 24-h expression of CD122 and NK1.1, and cytokine production after stimulation was assessed by flow cytometry. (A) Histograms show expression of CD122 and NK1.1 in gated iNKT cells infected (solid line) or not (dashed line) with MiY–T-bet or MiY-Runx3. Shown is one representative experiment out of three. (B) Bar graph shows the quantification of the three experiments. (C) Two-color histograms showing cytokine (IL-4 and IFN-g) production in gated iNKT cells from ET-2 mice infected or not with MiY–T-bet or MiY-Runx3. Shown is one representative experiment out of three. Bar graphs show the average and SEM of all the experiments (n = 3). Significance was assessed using a two-tailed unpaired t test. *p , 0.05. 5072 E PROTEIN ACTIVITY INFLUENCES iNKT SUBSET CHOICE bHLH domain of SCL/Tal1 does not mediate homodimerization tiation programs were so far unknown, and our results suggest that but has high affinity for E proteins, so ET2 competes with Id the level of E protein activity in developing iNKT cells regulates proteins to bind to endogenous E proteins. This approach allows the numbers of iNKT precursors that choose these alternative us to compete with both Id2 and Id3. Expression of ET2 results differentiation fates, rather than the canonical iNKT1 fate. in only a partial retention of E protein activity during iNKT cell Because in physiological conditions the level of Id induction development, as opposed to a complete knockout of Id proteins correlates with the intensity of the TCR signal, we hypothesize that that would maintain E-box binding activity at the high levels a similar mechanism could be responsible for this cell fate de- characteristic of DP thymocytes. This partial modulation is ad- termination, where iNKTs that receive stronger signals tend to vantageous to analyze the role of subtle differences in transcrip- become iNKT1s. This would be similar to early models of Th1 tion factor activity in cell fate decisions, especially for molecules versus Th2 differentiation (52, 53). However, like in Th differ- that exert some of their functions within a strict activity band, like entiation, other factors, including cytokines, are likely to play a E box proteins (30, 49). role in this fate decision. It is not clear at this point whether E This partial block in Id function results in a number of devel- proteins directly regulate the induction of GATA-3 and RORgT, or opmental alterations that highlight the many facets of development repress T-bet and Runx-3. An alternative model would posit that influenced by the careful balance of E and Id protein interactions. they control a currently uncharacterized checkpoint that deter- Although total iNKT cell numbers in the thymus are unaffected, mines which developmental pathway iNKTs choose. In support of there is a dramatic decrease in CD44+NK1.1+ (stage 3) mature this, we note that the levels of GATA-3, RORgT, or T-bet in the iNKT cells in the thymus, and the accumulation of a population different NKT subpopulations are almost indistinguishable be- of apparently immature (stage 2) iNKT cells that, after further tween NLC and ET-2 cells. The difference is how many cells have Downloaded from analysis, turn out to be iNKT2s and iNKT17s, recently described developed into each population in NLC mice versus in ET-2 mice, sublineages of iNKTs (13, 46, 47). In addition, ET2 expression which sustain E protein activity at higher levels during iNKT induces a profound defect in peripheral iNKT cells, suggesting differentiation. a defect in thymic exit. In conclusion, our results show that an alteration in the mag- Because total iNKT numbers in the thymus are normal, pro- nitude of the E protein activity during iNKT cell development

liferative expansion during development is clearly not impaired. promotes the iNKT2 and NKT17 programs, at the expense of the http://www.jimmunol.org/ The first wave of proliferation during iNKT cell development is iNKT1 program. myc controlled (50, 51). The second wave of proliferation happens during the stage 2 to stage 3 transition, and is IL-15 controlled for + Acknowledgments the conventional stage 3 NK1.1 NKTs (iNKT1) (41), but clearly We thank Dr. Bendelac, Dr. Sant’Angelo, and the National Institutes of there are alternative pathways because ET-2 NKTs do not express Health Tetramer Facility for providing reagents and P. Kincade for dis- CD122 (IL-2Rb). Candidates to mediate this second wave of pro- cussions. liferation for NKT2s are IL-17B or IL-25, but this remains to be tested. Disclosures by guest on September 28, 2021 Therefore, there is a profound defect in peripheral iNKT pop- The authors have no financial conflicts of interest. ulations, which our results suggest reflects a defect in thymic emigration because of the lack of Nrp upregulation in immature iNKTs. Although there could be other components to this defect, References we could not detect alterations in splenic iNKT cell survival, as 1. Gapin, L., J. L. Matsuda, C. D. Surh, and M. Kronenberg. 2001. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. described for liver iNKTs in Id2 knockout mice (19). Here again, Nat. Immunol. 2: 971–978. the difference between a full Id knockout and a partial Id inhi- 2. Godfrey, D. I., and S. P. Berzins. 2007. Control points in NKT-cell development. bition may explain these discrepancies. It is, however, important Nat. Rev. Immunol. 7: 505–518. 3. Matsuda, J. L., T. Mallevaey, J. Scott-Browne, and L. Gapin. 2008. 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