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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Dec 15, 2012.
Published in final edited form as:
PMCID: PMC3237743
NIHMSID: NIHMS333842

IL-15 regulates homeostasis and terminal maturation of NKT cells1

Abstract

Semi-invariant natural killer T (NKT) cells are thymus-derived innate lymphocytes that modulate microbial and tumour immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learnt regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-xL expression. Additionally, IL-15 regulated thymic developmental stage 2 (ST2) to ST3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C as well as several NK cell receptors in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-xL, and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

Introduction

Natural killer T (NKT) cells are thymus-derived lymphocytes whose functions are regulated by foreign or self glycolipid ligands presented by CD1d molecules. Most NKT cells recognise antigen with a semi-invariant T cell receptor (TCR) consisting of an invariant mouse Vα14Jα18 (human Vα24Jα18) α-chain that pairs with a Vβ8.2 (human Vβ11), Vβ7 or Vβ2 β-chain. Upon agonistic ligand recognition, NKT cells rapidly secrete large amounts of immunoregulatory cytokines and upregulate costimulatory molecules to initiate and modulate the effector functions of myeloid and lymphoid cells. By doing so, NKT cells regulate microbial and tumour immunity as well as autoimmune diseases (1, 2). NKT cells acquire these immunoregulatory properties during ontogeny through poorly understood processes. NKT cell frequency varies both between mouse strains – highest in BALB/c (1–2 million in thymus, liver and spleen) and the lowest in the autoimmune disease predisposed strains NOD and SJL (0.1–0.3 million)– and between healthy human individuals – ranging from 0.01–5.0% in peripheral blood (3). Hence, understanding how NKT cells develop, regulate their frequency and acquire functional potential is critical for designing NKT cell-based therapies.

NKT cells develop in the thymus and acquire markers typical of natural killer (NK) and activated/memory T cells as they mature (3). NKT cell precursors (CD24HICD69+CD44NK1; stage-0: ST0) emerge from CD4 and CD8 double-positive (DP) thymocytes soon after expression of the semi-invariant TCR in a process requiring three transcription factors: HEB–a basic helix-loop-helix E protein, RORγt–a retinoic acid orphan receptor, and c-Myb (47). Precursor ST0 cells proliferate extensively in a c-Myc-dependent manner (8, 9) and undergo positive selection through heterotypic interactions between the semi-invariant TCR and CD1d molecules expressed by DP thymocytes (1, 3). Further development and differentiation into ST1 (CD24CD44NK1.1), ST2 (CD24CD44+NK1.1) and to fully functional ST3 (CD24CD44+NK1.1+) NKT cells require transcriptional regulation by PLZF (10, 11), c-Myb (7), NF-κB/Rel-A (1215), Egr-2 (16), Gata-3 (1719) and T-bet (17, 20), as well as sustained homotypic interactions between the SLAM family members, Slamf1 and Slamf6 (21), by a mechanism involving the SAP and Fyn signaling module (2226). Furthermore, effector differentiation, which is ontogenetically coupled in NKT cells, is regulated by Itk (27), NF-κB (28), T-bet (20, 29), Egr-2 (16), Th-POK (30) and CSF-2 (31). Upon lineage commitment and the emergence of ST0 NKT precursors and through subsequent stages of differentiation and maturation, thymic and peripheral NKT cells are maintained by complex, yet poorly understood homeostatic mechanisms involving NF-κB (28), RelA (15), CYLD (32), c-Myc (8, 9), CD122 (33), γc (34), IL-7Rα (34), IL-15 (35, 36), IL-15Rα (37, 38), IRF-1 (39), calcineurin (16), Egr-2 (16) and Id2 (40).

During differentiation, NKT cells acquire the ability to produce IL-4 (ST1), IL-4 and IFN-γ (ST1and ST2) or IL-4LO and IFN-γHI (ST3) in response to TCR stimulation (27, 41), and secretion of these cytokines is regulated by CSF-2 (31). At the ST2 and ST3 stages, NKT cells also acquire memory T cell markers (CD44HI, CD62LLO, CCR7) as well as NK cell markers (NKG2D, Ly49 and NK1.1) and cytolytic function (granzyme B and perforin) (1, 42). Whilst a substantial fraction of ST2 cells remains in the thymus and matures to ST3, a small fraction egresses from the thymus and immigrates into the spleen and liver whereupon further maturation occurs (41, 43, 44). Migration to these peripheral sites depends on CXCR6 (45), whilst continued development and maintenance in the liver require LFA-1 (46) and Id2 (40). Even though mature thymic and splenic NKT cells are phenotypically similar, they behave as distinct functional populations at these sites because the developmental cues and perhaps the agonistic ligands are distinct at different sites (44, 47, 48). Thus, unalike conventional T lymphocytes, which undergo complete ontogenetic maturation in the thymus but commit to effector differentiation only upon antigen recognition in the periphery, NKT cells complete both these processes within and outside the thymus. Nonetheless, the instructive signals that differentially regulate the maturation and acquisition of effector functions of NKT cells remain poorly understood.

IL-15 is a pleiotropic cytokine that regulates the development and maintenance of several subsets of innate lymphocytes, including γδ T, CD8αα T, NK and NKT cells (49). Furthermore, IL-15 signals the ontogeny, effector differentiation and Mcl-1-dependent survival of NK cells (50). Moreover, thymic and peripheral NKT cells develop poorly in IL-15-deficient (IL-150) mice (35, 36). In the periphery, this is due to impaired NKT cell homeostatic proliferation (36). In the thymus, NKT cell proliferation appears intact, yet IL-150 mice develop a severe blockade in NKT cell ontogeny at ST2, with leaky progression to ST3 (36). What role IL-15 signals play during thymic NKT cell ontogeny and whether IL-15 is essential for NKT cell functional maturation in the periphery remain unknown.

Here we provide evidence that thymic and peripheral NKT cell survival critically depends on IL-15. One important downstream target of IL-15 appears to be Bcl-xL. Furthermore, we describe a new function for IL-15 by which it regulates ST2 to ST3 lineage progression and terminal NKT cell maturation, which is consistent with a role of T-bet in regulating ST2 to ST3 transition and terminal differentiation.

Materials and Methods

Mice

B6-IL-150, B6.129-IL-7α0, B6.J180 and B6.129-CD1dTD mice (34, 35, 5153) were generous gifts from J. Peschon (Immunex), M. Taniguchi (RIKEN, Japan) and A. Bendelac (U Chicago), respectively. B6-IL-150 and B6.129-IL-7Rα0 mice were bred with B6-Eμ36-Bcl-2tg (54) and C-Lck-Bcl-xLtg (55) mice. B6.C-IL-150;Bcl-xLtg mice were backcrossed to B6-IL-150 mice for 6 generations. All mouse crosses and experiments were performed under approved by Institutional Animal Care and Use Committee protocols.

Antibodies and Reagents

All antibodies used for the identification of NKT cells were from BD Pharmingen as described (56). Anti-BrdU-Alexa-647 (PRB-1; Invitrogen), anti-human Bcl-xL-PE and IgG3 isotype control-PE (7B25 and B10; Southern Biotech), anti-mouse Bcl-2-PE (3F11) and Armenian hamster IgG1κ isotype control-PE (A19-3), anti-human Bcl-2-PE (6C8) and Armenian hamster IgG2λ isotype control-PE (Ha4/8), anti-Mcl-1 rabbit polyclonal (Rockland), chicken anti-rabbit-Alexa-647 (Invitrogen), anti-T-bet-Alexa-647 (eBio4B10; eBiosciences) and mouse IgG1 isotype control-Alexa-647 (P3; eBioscience), anti-Gata-3-Alexa-647 (L50-823) and mouse IgG1κ isotype control-Alexa-647 (MOPC-21), anti-PLZF (D-9, Santa Cruz), mouse IgG1 isotype control (eBioscience), and anti-mouse IgG-FITC (eBiosciences) antibodies were purchased. α-galactosylceramide (αGalCer; KRN7000) was generously provided by Kirin Brewery Company (Gunma, Japan) or purchased from Funakoshi Co. Ltd. (Tokyo, Japan). Mouse αGalCer-loaded CD1d monomers were obtained from the NIH Tetramer Facility (Emory University). Preparation of αGalCer-loaded CD1d tetramer from monomer and their use are described elsewhere (56).

Flow Cytometry

Antibodies and staining procedures for the identification of NKT cells were as described elsewhere (56). NKT cells were identified as CD3ε+tetramer+ cells among B220LO splenocytes and hepatic mononuclear cells or CD8LO thymocytes. Four-colour flow cytometry was performed with a FACSCalibur instrument (Becton-Dickinson) whilst seven-colour flow cytometry was performed with a LSR-II instrument (Becton-Dickinson). Data were analyzed with FlowJo software (Treestar Inc.). Absolute NKT cell numbers were calculated from % tetramer+ cells and total number of cells recovered from each organ.

Ex Vivo Stimulation of Thymocytes and Splenocytes with IL-15

Thymocytes and splenocytes were stimulated in vitro with 100 ng/ml recombinant human (rh) IL-15 (Miltenyi) for 5 days. Cells were then collected and stained with specific mAb for intracellular expression of Bcl-2, Bcl-xL, and Mcl-1.

Cytokine Responses

Individual, age (6–10 weeks old)-matched mice were injected i.p. with 5 μg αGalCer or vehicle (0.1% Tween-20 in PBS) as controls. After 4 hrs, serum, splenocytes and hepatic mononuclear cells were prepared. Cells were stained with anti-CD3ε mAb, tetramer and anti-IFN-γ or anti-IL-4 mAb and analysed by flow cytometry. Sandwich ELISA was performed as described (56) to monitor serum cytokine response.

Cells were fixed and permeabilized with cytofix/cytoperm solution for intracellular staining or with cytofix/cytoperm plus (both from BD Pharmingen) for intra-nuclear BrdU staining according to the manufacturer’s protocol. Staining for intracellular IFN-γ and IL-4 also used GolgiStop (BD Pharmingen). Staining for intracellular T-bet, Gata-3, and PLZF used “Foxp3 staining buffer kit” (eBioscience). Intra-cellular and intra-nuclear staining was performed according to the manufacturer’s protocol.

Thymocyte sorting

Thymic NKT cells were enriched by magnetic sorting using a Pan-T cell isolation kit in combination with CD8 beads (Miltenyi) as instructed by the manufacturer. Enriched NKT cells from 5–10 mice were pooled and further purified by flow sorting CD3ε+tetramer+ cells (FACSAria; Becton-Dickinson). CD4 and CD8 double positive (DP) thymocytes were flow sorted as above. Freshly purified NKT cells and DP thymocytes were >98% pure as judged by CD3ε+tetramer+ and CD4/CD8 specific staining (data not shown).

RNA preparation, microarray hybridisation and analyses

Freshly purified NKT cells were washed and lysed by passage through a Qiashedder column (Qiagen). Total RNA was extracted per the manufacturer’s instructions (RNeasy, Qiagen). RNA yield was quantified spectrophotometrically (Nanodrop ND-1000) and aliquots electrophoresed to determine sample purity and concentration (Agilent 2100 Bioanalyzer). Microarray hybridizations were performed using One-colour hybridization on SurePrint G3 Mouse GE 60K Microarray (Agilent) by the Functional Genomics and Shared Resources Core (Vanderbilt University). Scanning was performed using a GenePix® 4000B Microarray Scanner (Molecular Devices). The resulting images were processed with Agilent Feature Extraction software to generate raw probe intensity data, which was subsequently normalised by the quantile method (57). Differential expression analysis was carried out with an empirical Bayes approach on linear model using limma package (58, 59). In this study, we defined differentially expressed genes as those whose log2 fold change values were ≥ +1.5 or ≥ −1.5 and whose nominal P-values were less than 0.001 (60). Next we performed the hierarchical clustering of the differentially expressed genes using the R AMAP Package (http://cran.r-project.org/web/packages/amap/). The microarray data are available at GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dtgxpkoosiokmpm&acc=); accession number: GSE32568.

Real time quantitative PCR

RNA was reverse transcribed with MuLV RT Reverse Transcriptase (Applied Biosystems) according to manufacturer’s protocol. qPCR was performed in the presence of IQ RealTime Sybr Green PCR Supermix and target gene-specific primers (Table S1) in an iQ5 thermocycler (Biorad). Results were analyzed using iQ5 software. Data were first normalized against β-actin control. As the CT values are obtained as log2 values, the normalised delta CT values of wt samples were subtracted from normalised delta CT values of IL150 samples. The resulting log2 delta delta CT values were transformed yielding relative expression, which enabled comparison between NKT cells isolated from the two strains. For this, IL150 sample, called the Calibrator was set to 1, and relative expression calculated as fold change in gene expression in wt NKT cells relative to the Calibrator.

TCRα re-arrangement

cDNA was prepared from total RNA isolated from DP thymocytes. qPCR for Vα14 to Jα18 rearrangement was performed as described above using the following forward (F) and reverse (R) primers: Vα14F (5′-AGGTCTTGTGTCCCTGACAG-3′), Jα18R (5′-CAGGTATGACAATCAGCTGAGTCC-3′), Vα8F (5′-TCACAGACAACAAGAGGACC-3′), and Jα5R (5′-AGTGAGCTGCCCCACAACCT-3′). PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.

Statistics

Comparisons of normally distributed continuous data were performed by one-way ANOVA with Tukey’s post test to determine significance. The significance of gene expression analyzed by qPCR was determined by unpaired T test. Statistical significance of Vα14-Jα18 rearrangement by qPCR was calculated by two-tailed, unpaired T-test. Comparisons were performed using GraphPad (Prism).

Results

IL-15 induces Bcl-2 family survival factors within thymic but not peripheral NKT cells

Previous reports demonstrated that NKT cell frequency is significantly lower in mice lacking IL-15 compared to wt mice due to altered NKT cell homeostasis within the thymus and spleen (35, 36). Whilst IL-15 regulates proliferation of splenic NKT cells, its role in homeostasis of the thymic subset has remained unclear (36). A re-evaluation confirmed that NKT cell frequency was altered in the absence of IL-15 and, hence, their numbers were reduced in the thymus, spleen and liver (Fig. 1A). Likewise, in vivo BrdU incorporation experiments revealed that splenic but not thymic NKT cell proliferation was impaired in IL-150 mice (Fig. 1B). These data indicate that, in addition to its role in cell proliferation, IL-15 plays another role(s) during thymic NKT cell ontogeny.

Figure 1
Defective NKT cell development and maintenance in IL-150 mice

Previous studies have suggested that IL-15 induces anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-xL and Mcl-1, to prevent apoptosis and to regulate homeostasis in vivo (49). For example, IL-15 promotes the survival of NK cells by inducing the survival factor Mcl-1 (50). An examination of the expression of the Bcl-2 family of survival factors by flow cytometry revealed that intracellular Bcl-2 expression was dramatically reduced in IL-150 thymic and splenic NKT cells (Fig. 1C). Although not as dramatic, intracellular Bcl-xL and Mcl-1 expression levels were also reduced either in thymic (Bcl-xL) or in both thymic and splenic (Mcl-1) IL-150 NKT cells when compared with wt NKT cells (Fig. 1C, D).

To test whether IL-15 induces survival factors in NKT cells, wt thymocytes and splenocytes were cultivated in the absence or presence of exogenous rhIL-15 for 5 days and Bcl-2, Bcl-xL and Mcl-1 expression in fresh or cultivated NKT cells (with or without rhIL-15) was evaluated using flow cytometry. Both fresh NKT cells and those cultivated without rhIL-15 were included as controls because expression of these survival factors changes during the 5d culture. The data revealed that IL-15 up-regulated the expression of Bcl-2 and Bcl-xL but not Mcl-1 in thymic NKT cells to levels above those expressed by freshly isolated NKT cells (Fig. 2A, B). Very few NKT cells cultivated without IL-15 expressed the three survival factors and, hence, when compared to this control, IL-15 also appeared to induce Mcl-1 expression in thymic NKT cells (Fig. 2B). IL-15 also modestly induced Bcl-2 and Bcl-xL expression within splenic NKT cells but not to the levels found in freshly isolated NKT cells; their expression was above control NKT cells cultured in medium without rhIL-15 (Fig. 2A, B). These data indicate that IL-15 has the potential to induce or maintain the expression of survival factors of the Bcl-2 family in thymic or splenic NKT cells, respectively, and, hence, to support NKT cell survival during development.

Figure 2
IL-15 up-regulates expression of the survival factors Bcl-2, Bcl-xL, and Mcl-1 within thymic and splenic NKT cells

Bcl-xL over expression in IL-150 mice supports NKT cell survival

We took a genetic approach to further investigate the role of IL-15-induced genes in the survival of thymic and peripheral NKT cells. Thus, Bcl-2 and Bcl-xl transgenes were independently introgressed into IL-150 mice. Analysis of the resulting mice at age 6–8 wks, when thymic and splenic cellularity is similar between the different strains, revealed that enforced Bcl-xL but not Bcl-2 over expression rescued the frequency and absolute numbers of thymic, splenic and hepatic NKT cells in IL-150 mice (Fig. 3A, B).

Figure 3
Bcl-xL over expression restores NKT cell development in IL-150 mice

Because the Bcl2 (Igμ) and Bclxl (pLck) transgenes are under the control of different promoters (54, 55), we assessed the expression patterns of both transgenes within different thymocyte subsets. The data revealed that both transgenes were expressed as early as the CD48 double negative stage of development, through the DP stage, and within CD4 and CD8 single positive thymocytes (Fig. 3C). Consistent with Bcl-2 expression during early thymocyte development, Bcl-2 transgene introgression into IL-7 receptor-α null mice, which poorly if at all develop conventional T and NKT cells (34, 61), rescued the development of both lineages (Fig. 3D). Therefore, the failure of Bcl-2 over expression to restore NKT cell numbers in IL-150 mice was not due to the lack of proper transgene expression or functionality, but most likely due to the fact that Bcl-2 plays a minor role when compared to Bcl-xL in conveying IL-15 signals for NKT cell survival. Together, the above data indicate that IL-15 relays specific, Bcl-xL-dependent survival signals during NKT cell ontogeny.

A previous study reported that IL-15-induced Mcl-1 specifically supported NK cell survival in vivo and in vitro (50) and that over expression of Bcl-2 in mice lacking IL-2Rβ – the common β-chain of IL-2 and IL-15 – receptors or in IL-150 mice repopulates NK cells in vivo (62, 63). Because NKT and NK cells are closely related innate lymphocyte lineages and because both lineages use IL-15 for survival, we ascertained whether IL-15 induces survival of the two cell types by similar mechanism(s). This question was addressed because Bcl-2 and Bcl-xL transgenes were expressed in NK cells from B6-Bcl-2tg and C-Bcl-xLtg mice (Fig. 3E). We found that, as previously reported (62, 63), Bcl-2 partially rescued hepatic but not splenic NK cells. Nonetheless, Bcl-xL over expression rescued NKT but not hepatic or splenic NK cells in IL-150 mice (Fig. 3F), which is consistent with the reported model in which NK cells predominantly utilize Mcl-1 for IL-15-induced survival (50). Thus, IL-15 appears to regulate survival of distinct lineages in a unique, cell type-specific manner, with Bcl-xL serving as an IL-15-induced survival factor for NKT cells.

Because Bcl-xL is required for DP thymocyte survival and Vα14 to Jα18 rearrangement (4), it is possible that IL-150 DP thymocytes poorly rearrange these distal gene segments, thereby resulting in low NKT cell numbers. To test this, Vα14 to Jα18 rearrangement within DP thymocytes flow sorted from wt, IL-150, Jα180 and CD1dTD – which rearrange Vα14 to Jα18 gene segments but are deficient in positive selection of NKT cells (52) – mice was quantified. No defect in the canonical Vα14 to Jα18 rearrangement was seen in IL-150 DP thymocytes (Fig. 4). Likewise, the more distal Vα8 to Jα5 rearrangement was also unaffected in IL-150 DP thymocytes as gauged by non-quantitative PCR (Fig. 4B). Interestingly, however, ~1.5-fold higher Vα14 to Jα18 rearrangement was observed in DP thymocytes of IL-150;Bcl-xLtg mice (Fig. 4A), which was statistically insignificant. This result suggests that any increase in the initial pool of NKT cell precursors in DP thymocytes of IL-150;Bcl-xLtg mice less likely led to increased numbers of NKT cells in these mice.

Figure 4
Normal TCRα rearrangement in IL-150 mice

IL-15 regulates terminal maturation of NKT cells

Because IL-15 deficiency reduced the numbers of thymic CD44NK1.1+ ST2 and CD44+NK1.1+ ST3 NKT cells, and because forced Bcl-xL over expression in IL-150 mice rescued total thymic and peripheral NKT cell numbers (Fig. 3), whether IL-15 has additional roles in NKT cell ontogeny was ascertained. Therefore, we determined whether IL-15 regulates terminal maturation of developing NKT cells as well, particularly at ST3 where NKT cells acquire most of their NK cell-like effector functions. Thus, the phenotype of IL-150;Bcl-xLtg NKT cells was compared to wt and IL-150 NKT cells. Surprisingly, akin to IL-150 NKT cells, phenotypic ST2 to ST3 transition, as measured by acquisition of the NK1.1 surface marker was still partially impaired in IL-150;Bcl-xLtg mice (Fig. 5A, B), despite near wt numbers of NKT cells in these mice. Additional phenotypic analyses revealed that expression patterns of few other mature NKT cell markers such as NKG2D, Ly6C, CD69, and Ly49C/I reflected those of thymic NKT cells that were blocked at ST2 (Fig. 5C) as these markers are predominantly expressed by NK1.1+ NKT cells (see Fig. 6A). Of those markers, the expression of Ly6C was also altered in hepatic, but not splenic NKT cells in IL-150 and IL-150;Bcl-xLtg mice (Fig. 5C). Thus, IL-15 is required not only for lineage survival but also for terminal maturation of NKT cells, especially of the thymic subset. Furthermore, the differential requirement for IL-15 in terminal differentiation (e.g., acquisition of Ly6C, NK1.1) of some (thymic and/or hepatic) but not all (splenic) NKT cells suggests that distinct signals instruct NKT cell maturation in distinct lymphoid organs.

Figure 5
IL-15 regulates terminal maturation of NKT cells
Figure 6
IL-15-induces Tbx21 and T-bet-regulated genes in developing NKT cells

IL-15 regulates multiple gene expression changes during ST2 to ST3 NKT cell transition

To define the mechanism by which IL-15 regulates terminal maturation of thymic NKT cells, microarray experiments were performed. For this, wt and IL-150 thymic NKT cells were flow sorted, and the derived RNA was subjected to hybridisation to a mouse microarray chip containing 60,000 genes; hybridisation was performed in duplicate (IL-150) or triplicate (wt). The microarray data are available at GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dtgxpkoosiokmpm&acc=); accession number: GSE32568. Select genes differentially expressed (minimally log2 fold change >+1.5/<−1.5 and nominal P-value <0.001) in wt and IL-150 NKT cells (Fig 6A) were then validated by quantitative (q), real-time PCR. We found that Tbx21 (T-bet) was up-regulated in wt NKT cells compared to IL-150 NKT cells (Fig. 6A, B). This finding is consistent with the previously reported IL-15-induced Tbx21-encoded T-bet expression in NKT cells in vitro (20). Furthermore, multiple Tbx21-regulated genes – such as Ifng, Gzma (granzyme A), Gzmc, Hopx (homeobox domain only containing protein x), and several NK cell receptor genes: Klra3 (NKG2E), Klrb1c (NK1.1), Klrc1 (NKG2A/B), and Klrk1 (NKG2D) (20, 29) – were up-regulated in wt thymic NKT cells, which predominantly consists of ST3 NKT cells (Fig 6B). Consistent with the lack of intracellular Bcl-2 in IL-150 NKT cells (Fig. 1C), the Bcl2 gene was poorly expressed in these cells (Fig. 6B). Conversely, Zbtb16 (PLZF), Tcf7 (TCF1) and Rorc (RORγt) genes, all signatures of ST1 and ST2 NKT cells were up-regulated in IL-150 thymic NKT cells (Fig. 6C), consistent with developmental arrest at ST2. Because T-bet is a critical regulator of differentiation in other lineages with cytotoxic properties (CTL and NK cells), our results are consistent with a role of Tbx21 in regulating ST2 to ST3 transition.

IL-15 regulates functional maturation of NKT cells

Terminally mature NKT cells produce the highest amounts of effector cytokines. Because phenotypically this subset of NKT cells appears impaired in IL-150 and IL-150;Bcl-xLtg mice, we determined whether they were functionally competent. For this purpose, the potent NKT cell ligand αGalCer (64) was administered i.p. into IL-15+/−, IL-150 and IL-150;Bcl-xLtg mice and 4 hrs later, intracellular and serum IL-4 and IFN-γ responses were monitored. Compared to IL-15+/− mice, significantly fewer splenic and hepatic NKT cells from IL-150 and IL-150;Bcl-xLtg mice expressed intracellular IL-4 and IFN-γ in response to in vivo stimulation (Fig. 7A, B). Furthermore, NKT cells that expressed IL-4 (splenic) and IFN-γ (splenic and hepatic) in IL-150 and IL-150;Bcl-xLtg mice, expressed these cytokines at significantly lower levels compared to those in IL-15+/− mice (Fig. 7A, B). Consistent with this expression pattern, the serum IFN-γ and IL-4 responses were also poor in IL-150 and IL-150;Bcl-xLtg mice compared to their IL-15+/− counterparts (Fig. 7C, D). Thus, IL-15 signalling is required for fully competent NKT cell responses in vivo.

Figure 7
IL-15 regulates functional maturation of NKT cells

IFN-γ and IL-4 responses by NKT cells require transcriptional regulation by T-bet and Gata-3 (17, 19). T-bet is also required for the terminal maturation of thymic NKT cells, as T-bet deficiency blocked NKT cell ontogeny at the ST2 to ST3 transition (20), similar to that seen with IL-15 deficiency (Fig. 5). Because Tbx21 expression is reduced in IL-150 NKT cells and IL-15 induces Tbx21 expression in NK cells in vitro (20), we investigated whether impaired NKT cell function in IL-150 and IL-150;Bcl-xLtg mice was due to altered levels of T-bet and Gata-3 expression. For this, thymic and splenic NK1.1 and NK1.1+ NKT cells from wt and IL-150 mice were probed for intracellular expression of T-bet and Gata-3. We found that the level of T-bet expression was reduced by 30% (splenic) to 50% (thymic) in IL-150 compared to wt NKT cells (Fig. 7E, G). Additionally, a slightly reduced percentage of thymic, but not splenic, NKT cells expressed T-bet in IL-150 mice. Similarly, intracellular Gata-3 expression was ~15–20% lower in thymic and splenic NK1 NKT cells but about similar in NK1+ NKT cells of IL-150 mice compared to their wt counterparts (Fig. 7F, G), consistent with somewhat more preserved IL-4 production in IL-150 NKT cells when compared to IFN-γ response. In wt NKT cells, Gata-3 expression level was constant, whilst T-bet levels increased with ST3 differentiation (Fig. 7G). Hence, the reduced T-bet expression (Fig. 7E) in IL-150 mice is consistent with reduced ST3 NKT cells in these mice. Thus, IL-15 signals are partially required for T-bet and Gata-3 expression in NKT cells and, possibly, for the robust IFN-γ and IL-4 cytokine responses seen in wt but not in IL-150 and IL-150;Bcl-xLtg mice.

Discussion

We describe three unique features of IL-15 that are essential for functional NKT cell development: One, it induces survival of thymic and peripheral NKT cells by regulating Bcl-xL and Bcl-2 expression. Two, IL-15 regulates T-bet expression and signals ST3 NKT cell induction and differentiation. And three, it regulates effector differentiation that is consistent the role of T-bet in this process. Thus, IL-15 plays a central role in NKT cell development and function.

It appears as though NKT cells narrowly escape death at each stage of their development: Immediately after positive selection they turn-on Nur77 expression in ST0 NKT cells (65), perhaps owing to high avidity interactions with their cognate ligand, viz., CD1d-self lipid complexes. In T cells, the transcription factor Nur77, in addition to inducing an apoptotic programme, when targeted to the mitochondrion binds to the otherwise anti-apoptotic factor Bcl-2, and by changing its conformation, converts it into a pro-apoptotic protein (66). Therefore, onward development of NKT cells perhaps requires survival signal(s) to protect the lineage from death. We have identified one such survival signal to be IL-15. It induces both Bcl-2 and Bcl-xL within thymic NKT cells in vitro. Yet, only genetic overexpression of Bcl-xL but not Bcl-2 confers survival potential to ST1 and ST2, but not ST3, NKT cells. These data are consistent with a previous report showing that enforced Bcl-2 expression does not rescue NKT cell development in IL-15Rβ-null mice (62). A recent report suggested that forced Bcl-2 overexpression or introgression of Bim-deficiency modestly rescues NKT cell development in the IL-15-presenting IL-15Rα-null mice (38). The differences in survival mechanisms in these three animal models currently remain unknown.

The global gene expression analysis of wt and IL-15-deficient NKT cells, as well as our published data (14), indicate that ST3 but not ST0 – 2 NKT cells express high levels of Bcl-2. Hence, it is possible that Bcl-2 functions as an important survival factor only upon commitment and development into ST3 NKT cells. Because the ST3 NKT cells are absent in IL-15-, IL-15Rα- and IL-15Rβ-null mice, another factor must be necessary to support NKT cell survival up to this stage. Our data suggest Bcl-xL provides that function. The failure of transgenic Bcl-2 to do so in this study perhaps reflects the need for an IL-15-induced, ST3-specific accessory factor to execute its function.

Previous studies have demonstrated that thymic and peripheral NKT cells represent distinct functional subsets (44, 47, 48). The factor(s) that impacts differentiation of these two subsets remained incompletely defined. We found that most thymic NKT cells in IL-150;Bcl-xLtg mice were blocked at the ST2-to-ST3 ontogenetic transition and lacked activation/memory marker expression as were IL-150 NKT cells. In striking contrast, splenic and hepatic NKT cells in IL-150 and IL-150;Bcl-xLtg mice underwent almost complete phenotypic maturation, suggesting that developmental cues in the thymus and periphery are somewhat distinct. In contrast, gene expression analyses indicated reduced T-bet expression in both thymic and splenic NKT cells of IL-150 mice. T-bet was previously shown to regulate the ST2-to-ST3 NKT ontogenetic transition (20, 29). Thus, reduced T-bet expression is consistent with the ST2-to-ST3 developmental block in IL-150 NKT cells. Furthermore, low levels of T-bet may in part explain the poor expression of NK cell receptors as well as the deficiency in ST3-specific IFN-γ response in IL-150 mice. Taken together, we conclude that IL-15 differentially impacts central and peripheral NKT cell maturation in vivo.

It is noteworthy that T-bet belongs to a group of transcription factors whose functions are regulated not only by their presence or absence but also by absolute levels. An example of such regulation is seen in effector CD8 T cell fate determination via graded expression of T-bet (67). Differentiation into effector and memory CD8 T cell fates was dictated by the levels of T-bet, where high T-bet expression induced an effector cell fate, whilst lower T-bet level directed cells into the memory pool. Such a model would in principle be consistent with a past report (38) and ours suggesting that partially reduced levels of T-bet expression in IL-150 mice could indeed significantly impair the NKT cell differentiation programme. Finally, because T-bet regulates expression of IL-15/IL-2Rβ (CD122) on NKT cells (this report and 29), and therefore IL-15 responsiveness, the two factors (IL-15 and T-bet) generate a positive feedback regulatory loop. The property of all such loops is that small changes in gene expression are amplified by self-sustained and self-amplified system oscillation. Therefore, we predict that the reduced but not the complete absence of T-bet observed in IL-150 NKT cells may be sufficient to prevent complete effector differentiation.

The importance of NKT cell function(s) is implicated in multiple infectious diseases. Because NKT cells acquire most of their NK cell-like properties at the last ST3 stage of their differentiation, and because IL-15 seems to regulate acquisition of these properties, we speculate that IL-15-encoded NKT cell functions will be important in controlling in vivo responses to infections. This will be an exciting area for future investigations. In conclusion, IL-15 functions by supporting survival of developing NKT cells and perhaps by inducing Tbx21 expression as a ST3-specific differentiation factor in NKT cells. NKT cells share this feature with NK cells and T cells, and are expected to collaborate with these lineages in executing Tbx21-induced functions in vivo.

Supplementary Material

Acknowledgments

We thank Drs. JJ Peschon (Immunex), M Taniguchi (RIKEN, Yokohama, Japan) and A Bendelac (U Chicago) for sharing B6-IL-150, B6.129-IL-7α0, B6-J180 and B6.129-CD1dTD mice; the NIH Tetramer Facility for unloaded and PBS57-loaded mouse CD1d monomers; and Kirin Brewery Company for KRN7000.

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