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of January 8, 2019. This information is current as Human Invariant NKT Cells Driven by IL-7 Innate-Like Effector Differentiation of Casorati Aiuti, Andreas Radbruch, Paolo Dellabona and Giulia Ferdinando Bombelli, Giovanna Borsellino, Alessandro Hyun-Dong Chang, Grazia Andolfi, Ulrike Benninghoff, Claudia de Lalla, Nicola Festuccia, Inka Albrecht, http://www.jimmunol.org/content/180/7/4415 doi: 10.4049/jimmunol.180.7.4415 2008; 180:4415-4424; ; J Immunol References http://www.jimmunol.org/content/180/7/4415.full#ref-list-1 , 22 of which you can access for free at: cites 45 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved. Copyright © 2008 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on January 8, 2019 http://www.jimmunol.org/ Downloaded from by guest on January 8, 2019 http://www.jimmunol.org/ Downloaded from

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Human Invariant NKT Cells Driven by IL-7Innate-Like Effector Differentiation of

CasoratiAiuti, Andreas Radbruch, Paolo Dellabona and Giulia Ferdinando Bombelli, Giovanna Borsellino, AlessandroHyun-Dong Chang, Grazia Andolfi, Ulrike Benninghoff, Claudia de Lalla, Nicola Festuccia, Inka Albrecht,

http://www.jimmunol.org/content/180/7/4415doi: 10.4049/jimmunol.180.7.4415

2008; 180:4415-4424; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/180/7/4415.full#ref-list-1

, 22 of which you can access for free at: cites 45 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Innate-Like Effector Differentiation of Human Invariant NKTCells Driven by IL-71

Claudia de Lalla,2* Nicola Festuccia,* Inka Albrecht,¶ Hyun-Dong Chang,¶ Grazia Andolfi,†

Ulrike Benninghoff,† Ferdinando Bombelli,‡ Giovanna Borsellino,§ Alessandro Aiuti,†

Andreas Radbruch,† Paolo Dellabona,2* and Giulia Casorati2*

Conventional MHC-restricted T lymphocytes leave thymus with a naive phenotype and require Ag-dependent stimulation coupledto proliferation to acquire effector functions. Invariant (i)NKT cells are a subset of T lymphocytes considered innate because theydisplay an effector memory phenotype independent of TCR stimulation by foreign Ags. We investigated the effector differentiationprogram followed by human iNKT cells by studying cells from a relevant set of fetal thymi and umbilical cord blood samples. Wefind that human fetal iNKT cells have already started a differentiation program that activates the epigenetic and transcriptionalcontrol of ifng and il4 genes, leading at birth to cells that express these cytokines upon TCR signaling but independently ofproliferation in vitro. Both ex vivo and in vitro analysis of fetal and neonatal iNKT cells delineate an effector differentiationprogram linked to cell division in vivo, and they identify IL-7 as one of the crucial signals driving this program in the apparentabsence of Ag stimulation. Consistent with these data, human fetal and neonatal iNKT cells are hyperresponsive in vitro to IL-7in comparison to conventional T cells, owing to an increased expression and signaling function of the IL-7 receptor �-chain. Theinnate nature of human iNKT cells could thus derive from lineage-specific developmental cues that selectively make these cellsefficient IL-7 responders following thymic selection. The Journal of Immunology, 2008, 180: 4415–4424.

C onventional MHC-restricted CD4� and CD8� T cells de-velop in the thymus and are released in the periphery asphenotypically and functionally naive lymphocytes,

which are devoid of effector functions with the exception of IL-2production (1, 2). The expression of diverse effector cytokinegenes by T cells requires coupling of activation, cell division, anddifferentiation, which are necessary to release epigenetic con-strains on cytokine genes and to activate transcription factors spe-cific for cytokine gene promoters (2, 3). This whole process istriggered by the cognate interaction of naive T cells with profes-sional APCs in secondary lymphoid organs, and it requires days tobe completed. As a result of priming, naive T cells undergo clonalexpansion and differentiate in effector memory T cells, which arecharacterized by a primed phenotype and rapid effector functionsupon Ag recall (2).

IFN-� and IL-4 are considered the paradigm of effector cyto-kines produced by primed T cells. The expression of ifng and il4genes depends on the activation/up-regulation of T-bet and GATA-3

transcription factors, respectively (4, 5). T-bet directly trans-activatesthe Ifng promoter and induces remodeling of the ifng locus (6).GATA-3 is responsible for the induction of chromatin remodeling atthe Th2 cytokine locus by acting on specific control sequences (7).

The expression of cytokine genes by T cells is also regulated byepigenetic modifications that determine the accessibility of tran-scription factors to the regulatory regions of their target genes (8).The epigenetic modification that involves DNA is methylation ofcytosine residues in the context of symmetrical CpG dinucleotides,which are enriched in DNA stretches called CpG islands (9). De-methylation of CpG islands is associated with gene transcription(10) and is facilitated by DNA synthesis and cell division (11).Agents that inhibit DNA methylation enhance IFN-� or IL-4 ex-pression, while the levels of DNA methylation in the cytokinepromoters correlate inversely with differences in cytokine expres-sion between naive and memory T cells, suggesting a role for DNAmethylation in the regulation of T cell function (12, 13).

CD1d-dependent invariant (i)NKT3 cells are a strikingly conservedsubset of T lymphocytes that recognize exogenous and endogenousglycolipid Ags presented by CD1d, and that are implicated in thecontrol of autoimmune, infectious, and tumor conditions (14, 15).iNKT cells are considered innate lymphocytes due to the expressionof an effector memory phenotype independent of TCR stimulation byknown foreign Ags. iNKT cells 1) homogeneously express theprimed-memory CD45R0 and CD44high markers in man and mouse,respectively; 2) express the differentiation marker CD161, the NKreceptor NKR-P1a-c; 3) rapidly (hours) produce IFN-� and IL-4 uponactivation both in vivo and ex vivo; and 4) are typically CD4� orCD4�CD8�.

*Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Pro-gram, DIBIT, San Raffaele Scientific Institute, †San Raffaele Telethon Institute forGene Therapy, ‡Division of Obstetrics and Gynecology, San Raffaele Scientific In-stitute, Milan; §Laboratory of Neuroimmunology, Fondazione Santa Lucia, Rome,Italy; and ¶Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany

Received for publication December 6, 2007. Accepted for publication January21, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 The study was supported by grants from the Italian Association for Cancer Re-search, Italian Ministry of Health Ricerca Finalizzata, and Fondazione Cassa diRisparmio delle Provincie Lombarde (to G.C. and P.D.), and in part by FondazioneTelethon (to A.A.).2 Address correspondence and reprint requests to Dr. Claudia de Lalla, Dr. PaoloDellabona, and Dr. Giulia Casorati, Experimental Immunology Unit, Cancer Immu-notherapy and Gene Therapy Program, DIBIT, San Raffaele Scientific Institute, viaOlgettina 58, 20132 Milan, Italy. E-mail addresses: [email protected],[email protected], and [email protected]

3 Abbreviations used in this paper: iNKT, invariant NKT; �GalCer, �-galactosyl cer-amide; AB, adult blood; CB, umbilical cord blood; CIRE, conserved intronic regu-latory element; FT, fetal thymus; TEM, effector memory T; TREC, TCR excisioncircle.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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Mouse iNKT cells develop in postnatal thymus and undergo alinear differentiation program consisting of proliferation, the up-regulation of the activation marker CD44, the acquisition of ef-fector functions beginning with IL-4 followed by IFN-�, and theexpression of the terminal differentiation marker NK1.1 (CD161)(16, 17). The final NK-differentiation step takes place in both thymusand periphery and is regulated by a number of molecules, includingIL-15, which plays a critical role in the establishment and homeostasisof the mature mouse NKT cell compartment (18, 19). Mouse iNKTcells colonize the periphery already armed with effector functions be-cause, unlike conventional T cells, they activate the expression of IL-4and IFN-� already in thymus by a process that involves both epige-netic derepression of the ifng and il4 cytokine genes and expression ofconstitutive cytokine transcripts (20, 21).

Unlike mouse iNKT cells, human iNKT cells can be detected infetal thymus between 12 and 20 wk of gestation (22) and persist inpostnatal thymus (23, 24). Human iNKT cells displaying the im-mature CD4�CD161� phenotype predominates in thymi of anyage and in neonatal blood (22–24). The immature iNKT cells un-dergo a postthymic maturation, leading to the appearance of ma-ture CD4�CD161� and CD4�CD161� iNKT cells present inadults.

Human iNKT cells exhibit at birth an intriguingly dissociatedphenotype: they display a primed/memory surface phenotype, but

no effector cytokine expression upon activation ex vivo (25), call-ing into question the truly innate nature of these lymphocytes. Wethus investigated thoroughly the effector competence of fetal andneonatal human iNKT cells in direct comparison with T cells. Wefind that human iNKT cells, unlike conventional T cells, have al-ready started an effector differentiation program in fetal life, con-sisting in the epigenetic derepression of il4 and ifng loci and in theactivation of GATA-3 and T-bet. Furthermore, we identify IL-7,and not IL-15, as a crucial signal driving the differentiation pro-gram of human iNKT cells.

Materials and MethodsDonors and tissues

Umbilical cord blood (CB) mononuclear cells were obtained after normaldelivery, and peripheral blood was obtained from adult healthy donors.Fetal thymi (FT) were obtained at the 20–23 gestational wk, in accordancewith the guidelines set forth by “L. Mangiagalli” Hospital ethical commit-tee of Milano, Italy. The collection and use of human material were ap-proved by the local ethical committee under investigational protocols, anda written informed consent was obtained from all of the tissue donors.

Preparation of PBMCs and thymocytes

Total mononuclear cells were separated from heparinized adult blood (AB)or CB by Ficoll-Hypaque (Pharmacia) density gradient centrifugation.

FIGURE 1. Neonatal iNKT cells produce cytokines upon in vitro stimulation independently of cell division. iNKT cells were identified by flowcytometry as CD3�V�24�V�11� cells among fetal thymocytes (FT) (20–23 wk), umbilical cord blood mononuclear cells (CB), or adult PBMCs (agerange 25–62 wk) (AB). A, Frequency of iNKT cells ex vivo was determined in FT (n � 11), CB (n � 41), and AB (n � 50). Data are reported as meanvalues � SD. B, Percentage of iNKT cells expressing CD161, CD4, and CD45RO ex vivo was examined in FT (n � 10), CB (n � 11), and AB (n � 10).Data are reported as mean values � SD. C, Frequency of iNKT cells from CB- (n � 3) and AB- (n � 10) producing intracellular IFN-� and IL-4 wasdetermined by flow cytometry upon activation with PMA/ionomycin (1 h) and brefeldin A (1.5 h) or stimulation with anti-CD3 � anti-CD28-coated beadsfor 12 h followed by PMA/ionomycin (1 h) and brefeldin A (1.5 h), as indicated and described in Materials and Methods. Data are reported as meanvalues � SD. D, CB-derived mononuclear cells were labeled with CFSE and cultured with anti-CD3 � anti-CD28-coated beads at 1:1 T cell-to-bead ratio.After 16 and 72 h, CFSE dilution profiles of neonatal iNKT and T cells were compared by flow cytometry analysis. Data are representative of threeindependent experiments. Statistically significant differences (p � 0.05) were calculated by t test and are indicated by � and #.

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Thymus specimens were homogenized on a cell strainer (Falcon) to obtaina single-cell suspension.

Isolation of iNKT cells and T cells

CB iNKT cells were sorted by staining with anti-V�11-FITC, anti-V�24-PE (26), and anti-CD3-APC (BD Biosciences). CB CD4� naiveT cells were sorted after staining as CD3�CD8�CD45RA T cells usinganti-CD8-PECy5, anti-CD45RA-FITC (both from BD Biosciences),and anti-CD3-allophycocyanin mAbs, together with anti-V�11-PE andanti-V�24-PE mAbs to gate out V�24�/V�11� iNKT cells. AdultCD4� and CD4� iNKT cells were sorted by staining with anti-CD3-allophycocyanin, anti-CD4-PECy7 (BD Biosciences), anti-V�24-PE,and anti-V�11-FITC. Adult CD4�CD3�CD45RA�CCR7� effectormemory T (TEM) cells (27) were sorted by staining with anti-CCR7 (BDBiosciences) followed by PE-labeled anti-mouse �-chain (SouthernBiotechnology Associates), anti-CD3-allophycocyanin, anti-CD45RA-FITC, anti-CD8-PECy5 (BD Biosciences), anti-V�24-biotin, anti-V�11-biotin, and streptavidin-PECy5 to gate out iNKT cells and CD8�

T cells. Purified iNKT and T cells were either immediately lysed for exvivo gene-expression analysis or stimulated with anti-CD3 � anti-CD28 beads as described below.

Flow cytometry analysis

iNKT cells were identified by staining with anti-V�24-PE or -biotin, anti-V�11-FITC or -PE, anti-CD3-APC, and streptavidin-PECy5. The expres-sion of surface markers was assessed with anti-CD161-PE, anti-CD4-PE,anti-CD45RO-PE, anti-CD122-biotin, streptavidin-PECy5 (all from BDBiosciences), and anti-CD127-PE (Beckman Coulter).

To evaluate cell division ex vivo, cells were stained with anti-CD3-allophycocyanin, anti-V�11-FITC, anti-V�24-biotin, and streptavidin-PECy5, and fixed/permeabilized and stained with anti-Ki67-PE mAb (BDBiosciences). Intracellular cytokines production was detected as described(28). Invariant TCR was stained with anti-CD3, anti-V�24, and anti-V�11Abs before and after cell pemeabilization to compensate for the TCRdown-regulation induced by the stimulation with PMA/ionomycin. Sam-ples (at least 106 T cells) were acquired on a FC 500 flow cytometry system(Beckman Coulter) and analyzed with FlowJo software (TreeStar).

Stimulation with anti-CD3 � anti-CD28 beads

Sorted iNKT or T cells were stimulated with anti-CD3 � anti-CD28-coatedbeads (Dynal Biotech) at a 1:3 T cell-to-bead ratio. Culture supernatants wereharvested at the indicated time points and completely replaced with fresh me-dium. The concentrations of secreted IFN-�, IL-4, and IL-2 were determinedwith a Th1/Th2 cytometric bead array kit (BD Biosciences).

For CFSE-based proliferation assays, CB mononuclear cells were la-beled with CFSE and stimulated with anti-CD3 � anti-CD28-coated beadsin culture medium at 37°C. Cells were harvested, stained with anti-V�24-biotin, streptavidin-PECy5, anti-V�11-PE, and anti-CD3 allophycocyaninmAbs and analyzed by flow cytometry.

For gene-expression analysis, purified iNKT and T cells were stimulatedwith anti-CD3 � anti-CD28-coated beads for 12 h, harvested, and lysed asdescribed below.

Quantitative RT-PCR

Total RNA was extracted from primary or activated iNKT and T cells withan additional DNA digestion step (RNase-Free DNase set; Qiagen) andreverse transcribed. Real-time PCR was performed on cDNA with theLightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics)using the following primers: UbcH5B sense 5�-TCTTGACAATTCATTTCCCAACAG-3�, antisense 5�-TCAGGCACTAAAGGATCATCTGG-3�; T-bet sense 5�-CCCCGGCTGCATATCG-3�, antisense 3�-ATCCTTTGGCAAAGGGGTTA-5�; IL-4 sense 5�-CGGCAGTTCTACAGCCACCAG-3�, antisense 5�-CCAACGTACTCTGGTTGGCTTC-3�; IFN-�sense 5�-CGAGATGACTTCGAAAAGCTG-3�, antisense 5�-ATATTGCAGGCAGGACAACC-3�, GATA-3 sense 5�-GAACCGGCCCCTCATTAAG-3�, antisense 5�-ATTTTTCGGTTTCTGGTCTGGAT-3� (29). Datawere evaluated using the LightCycler software version 3.5.28 (Roche Di-agnostics) and the second derivative maximum algorithm. Specificity of thePCR product was confirmed by melting curve analysis. Serial dilutions ofhuman CD4 cells were used to generate the standard curves.

Enrichment of IL-4-producing neonatal iNKT cells in vitro

CB mononuclear cells were expanded in vitro by culturing with 100 ng/ml�-galactosyl ceramide (�GalCer, Alexis) and rhIL-2 for 10 days at 37°C.

FIGURE 2. Human neonatal iNKT cells have al-ready activated the expression of effector cytokinegenes. iNKT (iNKT) and CD4� T (T) cells from CB (n �29), CD4� (iNKTCD4�), CD4� iNKT (iNKTCD4�),and CD4� TEM cells (TEM) from AB (n � 19) wereFACS sorted, as described in Materials and Methods.Defined numbers of sorted cells (purity � 95%) wereeither lysed immediately for RNA extraction or acti-vated with anti-CD3 � anti-CD28 mAb-coated beadsfor 12 h. Activated cells were lysed for RNA extraction,while their supernatants were pooled to determine theconcentration of secreted cytokines by cytometric beadarray. Reported cytokine concentrations were normal-ized for the number of stimulated cells. RNA extractedfrom each sorted iNKT or T cell subset ex vivo or afteractivation in vitro were pooled, and the expression lev-els of IL-4, GATA-3, IFN-�, and T-bet were evaluatedby quantitative RT-PCR. UbcH5B was used as an in-ternal control to normalize the PCR templates. A, IL-4cytokine secretion, IL-4, and GATA-3 mRNA level. B,IFN-� secretion, IFN-�, and T-bet mRNA level. Thevalues reported represent the natural mean of 29 CB and19 AB pooled samples, respectively.

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Cells were then activated with PMA/ionomycin for 2 h, stained for surfaceIL-4 (IL-4 secretion cell-enrichment and detection kit, Miltenyi Biotec),followed by anti-IL-4-PE, anti-V�11-FITC, and anti-CD3-PECy5.V�11�IL-4� iNKT cells were sorted and immediately frozen for CpGmethylation mapping.

Methylated CpG mapping of IL-4 gene

Methylation of CpG motifs was performed as described (30). The bisulfite-specific nested PCR was performed using the following primers: IL-4 pro-moter region outer sense 5�-gttatttgtatttggaagaagttagg-3�; outer antisense5�-TACCAAATAAATACTCACCTTCTACTC-3�; inner sense 5�-GATATTATTTTATTTTTTTTTAGGAGG-3�; inner antisense 5�-AAACTATTCAAAATTTTAATAATCTCC-3�; intronic region of conserved intronicregulatory element (CIRE) outer sense 5�-GGTATTATTTTTTTAGATGTTTTGGTG-3�; outer antisense 5�-TCCATTAAACAAATTTCTTATTCAAAC-3�; inner sense 5�-GGTGATGTTTTTAGTATTTTTAGGTATG-3�; inner antisense 5�-AATCTCCCCATTTACTAAAACAAAC-3�. ThePCR products were subcloned into a TA vector (Invitrogen), and individualclones were sequenced.

TCR excision circle (TREC) analysis

DNA was extracted from sorted fetal thymic and CB iNKT cells and Tcells. TREC number was determined by quantitative PCR using primersspecific for GADPH to normalize DNA content. The TREC sequences wereamplified using the primers sense 5�-CTGTCAACAAAGGTGATGCCACATCC-3�; antisense 5�-CCATGTCACACTGTGTTTTCCATCC-3�; probe 5�-(FAM)CTGCTCTTCATTCACCGTTCTCACG(TAMRA)p-3�. Genomic

GADPH was amplified using the primers sense 5�-ACCACAGTCCATGCCATCACT-3�, antisense 5�-GGCCATCACGCCACAGITT-3�;probe 5�-(FAM)CCACCCAGAAGACTGTGGATGGCC(TAMRA)p-3�. Astandard curve was prepared by serial dilutions of a plasmid containing theamplified TREC sequence. To ensure similar PCR conditions, the standardplasmid was diluted in genomic DNA extracted from a TREC-negative cellline. PCR analyses were performed on 100 ng of DNA using TaqMan Uni-versal PCR Master Mix (Applied Biosystems). The reactions were performedin an ABI Prism 7700 sequence detection system and data analyzed using theGeneAmp software (Applied Biosystems). The detection limits of this tech-nique are 3 TREC copies/100 ng of DNA.

Cell cultures with IL-7 in vitro

Total fetal thymocytes and purified T cells (Pan T Cell Isolation Kit II,Miltenyi Biotec) from CB and AB were cultured for 14 days at a concen-tration of 1 � 106 cells/ml in complete RPMI 1640 medium supplementedwith 10% normal human serum (Euroclone) in the presence of rhIL-7(Roche) or rhIL-15 (R&D Systems) at 20 ng/ml. Absolute numbers werederived from total cell counts, and iNKT and T cell frequencies were de-termined by flow cytometry. Intracellular cytokine production by iNKTand conventional T cells were determined as described above.

For CD161 expression analysis, CD3�CD161� T cells were sorted (purity�95%) by staining purified CB T cells with anti-CD161-biotin (Serotec) andstreptavidin-PECy5, and incubated with either rhIL-7 or rhIL-15 (20 ng/ml).After 7 days, cells were stained with anti-V�24-PE, anti-V�11-FITC, anti-CD161-biotin, and streptavidin-PECy5 and analyzed by flow cytometry.

FIGURE 3. il4 gene is already de-methylated in neonatal iNKT cells atcritical sites for transcription. A,Sorted CB iNKT (iNKT ex vivo) andnaive CD4�CD45RA� T (T) cellswere analyzed immediately (ex vivo)or, for iNKT cells, after expansion invitro with �GalCer and sorting of IL-4-producing cells (iNKT in vitro IL-4�) for the methylation status at theil4 gene promoter (nucleotide �66 to�412) and CIRE (nucleotide �542to �869) sequences by bisulfite-based cytosine methylation analysis.Black and white dots represent meth-ylated and demethylated CpG, re-spectively. B, The frequency of se-quenced chromosomes bearing ademethylated CpG at any given posi-tion in the il4 gene promoter andCIRE segments is reported for eachsorted cell type analyzed. The fre-quency is calculated by dividing thenumber of demethylated CpG at anygiven site by the number of total se-quenced chromosomes for eachsorted cell type.

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Stat-5 phosphorylation by IL-7

Purified CB-derived T cells were incubated at a concentration of 1 � 106

cells/ml with 0.8 ng/ml IL-7. After 60 min, cells were harvested, stainedwith anti-V�24-biotin, anti-V�11-FITC, anti-CD3-APC mAbs, andstreptavidin-PECy5, and then fixed, permeabilized with ice-cold methanolfor 10 min, and stained with anti-phospho-Stat-5 (Y694)-PE (BD Bio-sciences) at room temperature for 20 min. T cells were analyzed on aFACSCalibur cytometer (BD Biosciences).

ResultsHuman neonatal iNKT cells are poised to rapidly displayeffector functions

We first compared the effector phenotype and the functional com-petence of human iNKT cells from FT, CB, and AB (Fig. 1). First,human iNKT cells were present in 20–23-wk-old FT, where theyincreased 4-fold in frequency during the last trimester of gestationand less than 2-fold in the postnatal life (Fig. 1A), suggesting thatthe subset expanded substantially during fetal life and remainedessentially stable after birth. Second, in FT and CB, human iNKTcells display already a CD45R0 primed/memory phenotype, ac-companied however by an incomplete degree of NK differentia-tion, indicated by the low percentage of CD161 (Fig. 1B). Third,despite their primed/memory phenotype, human FT (data notshown) and neonatal iNKT cells were not fully armed with effectorfunctions ex vivo elicited by a brief (2.5 h) activation with PMA/ionomycin, unlike the adult ones (Fig. 1C). However, if a sustainedstimulation with anti-CD3 � anti-CD28-coated beads for 12 h pre-ceded the activation with PMA/ionomycin, a method shown tooptimize the detection of intracellular cytokines (31), a sizablefraction of iNKT cells became able to produce IFN-� and IL-4(Fig. 1C). This cytokine production did not require in vitro pro-liferation, as neonatal iNKT cells did not divide within 12 h of stim-ulation with anti-CD3 � anti-CD28, whereas they actively prolifer-ated after 72 h (Fig. 1D). Finally, both phenotypically immatureCD161� and mature CD161� iNKT cells activated in vitro with anti-CD3 � anti-CD28 beads followed by PMA/ionomycin exhibited acomparable production of IFN-� and IL-4 (data not shown).

These data show that neonatal iNKT cells are poised to rapidly displayeffector functions at birth independently of cell division in vitro.

Distinct transcriptional control of cytokine gene expression inneonatal iNKT and conventional T cells

In light of the above findings, we undertook a comparative molecularanalysis of the transcriptional control of ifng and il4 gene expressionin iNKT and T cells from CB and AB. Human neonatal iNKT cellsare mostly CD4�, while adult iNKT cells are subdivided into CD4�

and CD4� subsets and display a TEM phenotype (25). iNKT andCD4� T cells were therefore isolated from CB, while CD4� iNKT,CD4� iNKT, and CD4� TEM cells (27) were sorted from AB. Thesecells were directly analyzed ex vivo, or after 12 h of activation in vitrowith anti-CD3 � anti-CD28-coated beads, for the ability to secreteIL-4 and IFN-� and for quantitative expression of IL-4, IFN-�,GATA-3, and T-bet mRNA by real-time PCR.

As shown in Fig. 2A, neonatal iNKT cells, but not CD4� T cells,produced IL-4 upon TCR � CD28 activation in vitro. The secre-tion of IL-4 by neonatal iNKT cells upon activation was evenhigher than that of both adult CD4� and CD4� iNKT subsets.Consistently, neonatal iNKT, but not T cells, expressed detectableconstitutive IL-4 transcripts, which were further up-regulated uponTCR � CD28 stimulation. Both CD4� and CD4� adult iNKTcells maintained a level of constitutive IL-4 transcript similar tothat of neonatal iNKT cells; however, their capacity to up-regulateIL-4 mRNA levels upon TCR � CD28 stimulation was markedlyreduced as compared with that of neonatal iNKT cells, which was

particularly clear in the CD4� subset. Conventional T cells, incontrast, acquired the competence to produce IL-4 in vitro onlyupon differentiation, as shown by adult TEM cells; these cells, how-ever, did not show detectable constitutive IL-4 mRNA, but ex-pressed it only upon activation. Constitutive GATA-3 mRNA ex-pression was markedly higher in neonatal iNKT than in T cells.The constitutive GATA-3 expression increased progressively fromneonatal to adult iNKT cells and from naive T to TEM cells. Uponactivation, the expression of GATA-3 was maintained at the samelevel only in neonatal iNKT cells, whereas it was down-regulatedin the adult iNKT cells and in all T cell subsets analyzed.

As shown in Fig. 2B, neonatal iNKT cells, and not T cells,secreted IFN-� upon sustained TCR � CD28 activation in vitro.At variance with IL-4 production, adult CD4� and, to a lesserextent, CD4� iNKT cells secreted more IFN-� than did neonataliNKT cells, resulting in a shift of the cytokine balance from anIL-4 � IFN-� pattern displayed at birth, to a marked IFN-� � IL-4pattern displayed in adult life. In line with the cytokine secretion,neonatal iNKT and not T cells expressed detectable constitutiveIFN-� transcripts and up-regulated it markedly upon activation.Constitutive expression of T-bet mRNA was detected in neonataliNKT and not in T cells, while TCR-CD28 activation up-regulatedT-bet transcription in both cell subsets, even though the latter cellsdid not express IFN-�, suggesting that factors other than T-betmight be required for IFN-� production in naive T cells. Consistentwith the age-related shift toward a Th1 pattern, both constitutiveand inducible IFN-� and T-bet mRNAs increased from neonatal toadult iNKT cells, particularly within the CD4� subset. In analogywith IL-4, T cells acquired the production of IFN-� only afterbirth, upon differentiation from naive to TEM cells, which wasassociated with the appearance of both constitutive and activation-inducible transcription of IFN-� and T-bet.

FIGURE 4. iNKT cells divide more than T cells during fetal life. A,iNKT cells or T cells were FACS sorted from FT (n � 7) and CB (n � 6).The TREC number was determined in each sample as described in Mate-rials and Methods. Statistically significant differences (p � 0.01) werecalculated by t test and are indicated by # and �. B, Frequency of Ki67expression ex vivo evaluated in iNKT cells and T cells from FT (n � 5)and CB (n � 5) by flow cytometry. Numbers above the histogram barsindicate the mean values. Statistically significant differences (p � 0.05)were calculated by t test and are indicated by �.

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Together, these findings indicate that iNKT cells have already ac-tivated the transcription of both ifng and il4 loci during fetal life, andthey argue for a distinct regulation of IFN-� and IL-4 gene expressionduring fetal development between iNKT and conventional T cells.Furthermore, these findings also show a different regulation of ifngand il4 gene expression in neonatal vs adult iNKT cells.

Epigenetic derepression of effector cytokine genes in neonataliNKT cells

We next determined whether the distinct effector competence ofneonatal iNKT cells could be attributed also to epigenetic modi-

fications in the effector cytokine loci already present at birth. iNKTand CD4� T cells were purified from CB and subjected to bisul-fite-based cytosine methylation analysis to determine the degreeof demethylation at critical CpG residues in the promoter andthe CIRE of the human il4 gene. These two regions are impli-cated in the transcriptional regulation of this cytokine (32). Asshown in Fig. 3, A and B, CpGs in the il4 promoter and CIREshowed a higher degree of demethylation in the chromosomessequenced ex vivo from neonatal iNKT than from CD4� Tcells. The promoter was less methylated than was the CIRE,consistent with the 5�-to-3� direction of gene demethylation.

FIGURE 5. IL-7 recapitulates in vitro the innate-like human iNKT cell differentiation program. A, Frequency of iNKT and T cells expressing CD127and CD122 ex vivo determined by flow cytometry in FT (n � 5) and CB (n � 6). Reported are the mean values � SD. B, Representative dot plot analysisof CD127 expression in iNKT and T cells gated among CD3high thymocytes (FT) and CD3� CB-derived cells. C, Absolute numbers of iNKT and T cellsfrom FT and CB mononuclear cells determined ex vivo and upon 14 days of culture with IL-7. Shown is one representative experiment of three experimentsperformed. Numbers above the histograms indicate fold increase values calculated as ratio of cell numbers after culture with IL-7 vs ex vivo. D, CD161up-regulation by sorted neonatal CD161� iNKT and T cells (purity �95%) evaluated by flow cytometry upon 7 days of culture with IL-7. Data are themean values �SD of three independent experiments. E, Frequency of iNKT and T cells from FT- and CB-producing intracellular IFN-� and IL-4 wasevaluated by flow cytometry ex vivo and following 14 days of culture with IL-7. Cells obtained ex vivo or after culture in vitro were activated withPMA/ionomycin and brefeldin A for 2.5 h, fixed-permeabilized, and stained with cytokine-specific mAbs. Shown are the mean values � SD of threeindependent experiments. F, Representative dot plot analysis of intracellular IFN-� and IL-4 production by FT- and CB-derived iNKT cells upon culturein vitro with IL-7 as described in E. Statistically significant differences (p � 0.05) were calculated by t test and are indicated by �.

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The frequency of demethylated il4 genes in primary CB iNKTcells was consistent with that of iNKT cells competent to se-crete cytokines at birth upon TCR � CD28 activation. The de-gree of demethylation of il4 promoter and CIRE regions increasedfurther in neonatal iNKT cells enriched for IL-4 secretion afterexpansion in vitro for 10 days with the specific Ag �GalCer, con-firming the importance of the demethylation of these regulatorysequences for il4 gene expression. The differential methylation sta-tus of ifng and il4 loci in CB T and iNKT cells was further sub-stantiated in activation experiments using anti-CD3 � anti-CD28-coated beads for 2 days in the presence or absence of thedemethylating agent 5�-azacytidine (data not shown). The increasein the frequency of cells expressing IFN-� or IL-4 induced by5�-azacytidine was substantially lower in iNKT than in T cells,suggesting a higher basal level of demethylation at the ifng and il4genes in iNKT cells.

Collectively, these results demonstrate that human iNKT cellshave programmed the epigenetic derepression of effector cytokineloci already in fetal life, and they argue for a differential develop-mentally regulated epigenetic control of cytokine gene expressionbetween iNKT and T cells.

Human iNKT cells undergo cell division in vivo duringfetal life

Cell division in vivo during fetal life could favor the epigeneticderepression of effector cytokine genes found in neonatal iNKT

cells, and explain the expansion of the subset. We thereforedetermined the proliferative history of iNKT cells during fetallife. Conventional T and iNKT cells were sorted from fetalthymus and CB, and their TREC contents were determined byquantitative real-time PCR. As shown in Fig. 4A, fetal thymic Tand iNKT cells contained similar quantities of TRECs, suggest-ing comparable cell division in fetal thymus. CB iNKT cellscontained less TRECs than did FT iNKT cells, compatible withfour divisions during fetal life. CB T cells also contained fewerTRECs than did fetal thymic T cells, compatible with two celldivisions during fetal life and in line with published data (33).In CB, TREC content in iNKT cells was significantly lowerthan in T cells, indicating that iNKT cells undergo more celldivisions than do T cells during fetal life.

To assess whether iNKT cells were actively proliferating in FTor CB, iNKT and T cells from both of these compartments werestained for the intracellular expression of the cell cycle-relatedprotein Ki67 (34). Fig. 4B shows that up to 30% of both iNKT orT cells are cycling in the fetal thymus. The frequency of activelycycling iNKT cells and T cells decreases markedly in the CB;nevertheless, the low fraction of Ki67-expressing iNKT cells re-mains significantly higher than in T cells.

Thus, human iNKT cells divide more than do conventional Tcells during fetal life and become quiescent at birth, even thoughsome degree of slow turnover seems to persist throughout life.

FIGURE 6. Neonatal iNKT cells are hyperresponsive to IL-7. A, Expression of CD127 in primary CB iNKT and T cells was assessed ex vivo byflow cytometry. Indicated are the mean fluorescence intensity values � SD of five independent experiments. B, Frequency of CB iNKT and T cellsexhibiting Stat-5 phosphorylation upon culture with 0.8 ng/ml IL-7 for 60 min, determined by intracellular flow cytometry as described in Materialsand Methods. Shown are the mean values � SD of five independent experiments. C, Representative histograms of pStat-5 expression profile of CBiNKT and conventional T cells upon culture with medium alone (as negative control) or with IL-7 as indicated in B. In the absence of IL-7, pStat-5mean fluorescence intensity values were comparable for iNKT and conventional T cells. D, Frequency of neonatal iNKT cells producing intracellularIFN-� or IL-4 ex vivo or upon 24 h culture with IL-7 or medium alone as indicated. Cells obtained ex vivo or after culture in vitro were activatedwith PMA/ionomycin and brefeldin A for 2.5 h, fixed-permeabilized, and stained with cytokine-specific mAbs. Shown are the mean values � SDof three independent experiments. E, Representative dot plot analysis of IFN-� and IL-4 intracellular production by neonatal iNKT cells upon culturewith medium alone or IL-7 as described in D.

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IL-7 recapitulates human iNKT cellular differentiation programin vitro

IL-15, and to a lesser extent IL-7, plays an essential role in con-trolling the maturation and size of murine iNKT cells (18, 19). Weasked whether these cytokines could drive cell division, effectorcytokine expression, and NK differentiation in human fetal andneonatal iNKT cells in vitro. We first verified the expression ofIL-7R� (CD127) and IL-2/15R� (CD122) in FT- and CB-derivediNKT and T cells. Among CD3high fetal thymocytes, substantiallymore iNKT cells expressed CD127 compared with T cells (Fig. 5,A and B). CD127 became expressed by almost all iNKT and T cellsin CB. At variance with CD127, however, the expression ofCD122 was infrequent among FT- and CB-derived iNKT and Tcells.

Consistent with the distribution of receptors, iNKT cells fromthe two compartments expanded vigorously in response to IL-7 invitro (Fig. 5C), but not in response to IL-15 (not shown). Thegrowth of fetal thymic and neonatal T cells in response to IL-7 invitro was markedly lower than that of iNKT cells.

iNKT or T cell proliferation induced by IL-7 in vitro was con-firmed by measuring intracellular expression of Ki67 or by deter-mining CFSE dilution (data not shown). IL-7 up-regulated in vitrothe expression of CD161 in sorted CD161� neonatal iNKT cells,but not in T cells, indicating the capacity of the cytokine to selec-tively induce the NK differentiation in human neonatal iNKT cells(Fig. 5D). Finally, culturing cells with IL-7 for 14 days induced invitro the production of both IFN-� and IL-4 in a higher proportionof FT- and CB-derived iNKT cells than T cells (Fig. 5, E and F).The experiments described in Fig. 5 were carried out with eithertotal CB mononuclear cells or purified total T lymphocytes withoutsignificant differences (not shown).

Collectively, therefore, these results show that IL-7 recapitulatesin vitro the differentiation program followed by human iNKT cellsduring fetal and, possibly, postnatal life.

Neonatal iNKT cells are hyperresponsive to IL-7

Although similar fractions of iNKT and T cells from the CB ex-pressed CD127, iNKT cells grew and differentiated much moreefficiently than T cells in response to IL-7 in vitro, suggesting ahigher responsiveness of neonatal iNKT cells to this cytokine. In-deed, the mean fluorescence intensity of CD127 was significantlyhigher in CB iNKT than T cells, as shown in Fig. 6A. Furthermore,most of CB iNKT cells but only 50% of T cells responded to IL-7signaling by phosphorylating Stat-5 (35), indicating an increasedsignaling via the IL-7/CD127 pathway in human iNKT cells atbirth (Fig. 6, B and C). Remarkably, as shown in Fig. 6, D and E,IL-7 signaling triggered also the expression of intracellular IFN-�and IL-4 in 20% of CB iNKT cells, but not in T cells, as earlyas after 24 h of culture and independently of cell division (data notshown). Together, these results strongly argue for a major role forIL-7 in driving the proliferation, acquisition of effector functions,and NK differentiation of human fetal and neonatal iNKT cells invivo, due to a higher membrane expression of IL-7R�, resulting inturn in a stronger signaling cascade that leads to Stat-5phosphorylation.

DiscussionThis study shows that a sizable fraction (up to 20%) of humaniNKT cells is competent at birth for the production of IFN-� andIL-4 independently of cell division in vitro, and it displays a mo-lecular setting for effector cytokine gene expression that is alreadysimilar to that of adult iNKT cells. We provide ex vivo and in vitro

evidence that IL-7 is one of the signals crucial to drive the innate-like differentiation program of human iNKT cells in fetal life.

In mice, the IL-15 signaling pathway plays an essential role inthe maturation and overall population size of iNKT cells in thethymus and periphery (18, 19). CD122 is indeed expressed at lowlevels already in thymic immature CD44�NK1.1� iNKT cell pre-cursors, and it becomes progressively up-regulated upon subse-quent differentiation into intermediate CD44�NK1.1� and thenfully mature CD44�NK1.1� cells (18). Moreover, CD122 expres-sion is maintained high in all peripheral mature mouse iNKT cells,irrespective of CD4 coreceptor expression (18). In contrast,CD127 is expressed similarly by mouse iNKT cells at any matu-ration stage; however, IL-7 seems critical for the development ofiNKT cells, but it only plays a minor role in regulating the matu-ration and homeostasis of these cells (18). Our data show that onlyvery small fractions of human fetal and neonatal iNKT cells ex-press CD122, whereas they homogenously express CD127 andrespond vigorously to IL-7 in vitro by proliferating and differen-tiating to mature CD161� effector cells. Therefore, IL-7 dominatesthe human iNKT cell differentiation process during the fetal andperinatal life.

IL-7, however, might also play a functional role for humaniNKT cells in adult life. In adult humans, in fact, CD127 is ex-pressed by most iNKT cells, which grow in vitro in response to thecytokine, whereas CD122 is expressed only by a minor fraction ofthese cells, mainly in the CD4� subset, implying a selective andage-specific role for IL-15 in the expansion/homeostasis of thehuman CD4� iNKT cell subset ((23) and C. de Lalla, unpublisheddata). Overall, therefore, the control of maturation and size ofiNKT cells would seem to be differently regulated by IL-7 andIL-15 in humans and mice.

We showed that fetal and neonatal iNKT cells are hyperrespon-sive to IL-7 in vitro compared with T cells due to the higher den-sity of membrane IL-7R�, which is associated with a strongerIL-7-dependent signaling. We propose that the high level ofCD127 expression by human fetal iNKT cells is acquired in alineage-dependent manner during the thymic selection, driven byunique molecular interactions between developing iNKT cells andDP thymocytes (15, 36). These characteristics would allow iNKTcells to outcompete T cells for limiting concentration of IL-7 invivo, thereby explaining the greater proliferation and differentia-tion stage attained by iNKT cells in fetal life.

Human iNKT cells have indeed divided in vivo during fetal lifesignificantly more than have conventional T cells. This is compat-ible with the 4-fold expansion of iNKT cells from fetal thymus toumbilical cord blood that we document in this study. The expres-sion of Ki67 indicates that iNKT cells divide actively in fetal thy-mus; nevertheless, we cannot rule out that iNKT cell division con-tinues in the periphery of the the fetus, for example, in secondarylymphoid organs where IL-7 is available (37).

The IL-7-driven signaling and proliferation of human iNKTcells during fetal life should facilitate the epigenetic derepressionof effector cytokine loci, consistent with the role of DNA synthesiscoupled with cell cycle in the remodeling of cytokine loci in Tcells (2, 3). The opening of chromatin at effector cytokine loci infetal iNKT cells is linked to the early activation of T-bet andGATA-3 and the expression of constitutive transcripts coding forIFN-� and IL-4. Together, these molecular features allow humaniNKT cells, unlike conventional naive T cells, to express cytokinesat birth in the absence of cell division. T-bet and GATA-3 areinduced in conventional naive T cells by TCR signaling and aresustained by the IFN-�/Stat-1 and the IL-4/Stat-6 signal transduc-tion pathways, respectively (6, 7). iNKT cells are normal in micedeficient in the IFN-�/Stat-1 signaling axes (38), while Stat-6

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seems dispensable for IL-4 expression by iNKT cells (39), sug-gesting that the expression of T-bet and GATA-3 in this subsetmight be differentially regulated in comparison to naive T cells.How the two master gene regulators of Th1 and Th2 cytokines areactivated in developing human iNKT cells is therefore an openquestion.

Given the activated transcriptional machinery at cytokine loci, itis unclear why human neonatal iNKT cells require a sustainedTCR signaling (12 h) to secrete IFN-� and IL-4, in comparison toadult iNKT cells. It is possible that a sustained TCR signalingserves to increase the number of cytokine mRNA molecules abovea critical threshold for translation by inducing more transcriptionof cytokine genes, or to couple preexistent cytokine transcripts tothe translation machinery via integrated stress response (40),or both.

Mouse thymic immature CD44highCD161� iNKT cells produceIL-4 and progress to a prevalent IFN-� production upon differen-tiating into the most mature CD44highCD161� cells (16, 17).IFN-� and IL-4 expression seems always coupled in human NKTcells as early as we could assess in development, even though mostof these cells display at birth an immature CD161� phenotype.Nevertheless, human iNKT cells clearly modify their relative IL-4and IFN-� balance upon ageing by essentially increasing IFN-�production, suggesting that a differentiation-dependent modifica-tion of the cytokine balance may occur in human iNKT cells aswell. Human neonatal iNKT cells up-regulate GATA-3 upon TCR-dependent activation, whereas adult iNKT and CD4� TEM cellsdown-regulate it (31, 41), suggesting that GATA-3 expression isdifferentially regulated in iNKT cells early in their life. The main-tenance of high GATA-3 levels in neonatal iNKT cells might alsoaccount for their more pronounced Th2-like cytokine profile incomparison with the adult cells (31).

Our data show that culturing in vitro human neonatal immatureiNKT cells with IL-7 induces proliferation and increases the fre-quency of cells expressing CD161 and effector cytokines, recapit-ulating such effects in vivo. Interestingly, IL-7 can also rapidly (12h) trigger in vitro the expression of IFN-� and IL-4, independentlyof cell division, in a fraction of neonatal iNKT cells that are al-ready competent for cytokine expression. IL-7 is produced in sev-eral inflammatory conditions, such as arthritis (42), where it mighttrigger the secretion of effector cytokines by infiltrating iNKTcells, which could take part in the regulation of the pathologicalprocess.

In mice, the complete acquisition of the terminal NK differen-tiation marker CD161 by iNKT cells is stimulated by the expres-sion of CD1d in the periphery, most likely by MHC class II�

APCs (43–45). It is also possible that human iNKT cells completetheir phenotypic and functional differentiation by integrating bothself-Ag- and IL-7-induced signals in the periphery.

Overall, this study shows that human iNKT cells follow lineage-specific developmental cues, leading to the activation of IL-7 re-ceptor signaling and the distinct effector memory phenotype atbirth. Human iNKT cells, however, have not yet completely un-folded their effector memory functions at birth. It may take moretime after birth to complete the differentiation program, and thismay be linked to low-grade peripheral cell division upon IL-7signaling and, possibly, CD1d self-recognition.

AcknowledgmentsWe thank Dr. Sergio Abrignani for the critical reading of the manuscript.Drs A. Colombo and F. Ficara are acknowledged for collection and prep-aration of human fetal thymus.

DisclosuresThe authors have no financial conflicts of interest.

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4424 EFFECTOR DIFFERENTIATION OF HUMAN iNKT CELLS

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