of February 28, 2013.This information is current as

by Dendritic Cell Tuning of SHP-1Functional Education of Invariant NKT Cells

Dellabona and Giulia CasoratiLehuen, David Voehringer, H. Robson MacDonald, Paolo Anna Napolitano, Paola Pittoni, Lucie Beaudoin, Agnes

ol.1203466http://www.jimmunol.org/content/early/2013/02/20/jimmun

published online 20 February 2013J Immunol 

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The Journal of Immunology

Functional Education of Invariant NKT Cells by DendriticCell Tuning of SHP-1

Anna Napolitano,* Paola Pittoni,*,1 Lucie Beaudoin,† Agnes Lehuen,† David Voehringer,‡

H. Robson MacDonald,x Paolo Dellabona,* and Giulia Casorati*

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by

bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to

maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymo-

cytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells.

We hypothesized that iNKT cells require functional education by CD1d+ cells other than thymocytes to set their correct respon-

siveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly

reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant

mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring

nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras

revealed that CD1d+ dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic

emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity. The Journal

of Immunology, 2013, 190: 000–000.

Invariant NKT (iNKT) cells are a conserved subset of Tlymphocytes that express an invariant TCR (iTCR) a-chain(Va14-Ja18 in mice) paired with a restricted set of Vb genes

(Vb8.2, Vb7, or Vb2 in mice) (1). This TCR is restricted forthe MHC-like molecule CD1d. iNKT cells are prototypical“innate lymphocytes” that acquire effector-memory character-istics in the thymus and display effector functions without priorAg sensitization (1). iNKT cells undergo a unique developmentalprogram, in which CD4+CD8+ “double-positive” (DP) precursorsare positively selected by CD1d-expressing DP thymocytes (1).Postselection iNKT cells (stage 0 CD24hiCD44loNK1.12) undergosubsequent intrathymic differentiation into naive (stage 1, CD44lo

NK1.12) and Ag-experienced (stage 2, CD44hiNK1.12) cells (1).

Stage 2 iNKT cells expand dramatically and undergo the final NKmaturation (stage 3 CD44hiNK1.1+) in thymus and in periphery (1).iNKT cells recognize lipid Ags derived from several pathogenic

bacteria (2, 3), but are also potently autoreactive against CD1d-

expressing APCs that present stress-related endogenous lipid Ags

(4–6). Activated iNKT cells rapidly produce different cytokines and

license APC functions, which, in turn, stimulate both innate and

adaptive immune responses, promoting the control of infectious

pathogens, tumors, and autoimmunity (1).Because of their strong autoreactivity, iNKT cell responsiveness

requires regulation tomaintain effective host defensewhile preventing

hyperstimulation and, possibly, autoimmunity. Upregulation of ag-

onist self-lipids by APCs under stress/inflammation is onemechanism

to limit iNKT cell activation in normal conditions (4–6). It is unclear,

however, how the iNKT cell activation threshold is physiologically

tuned to lipid Ags generated by microbial infections and stress.

iNKT cells developing in transgenic (tg) mice expressing human or

murine CD1d only on thymocytes display increased Ag reactivity (7,

8), suggesting that iNKT cells undergo “functional education” to

establish TCR-responsiveness by interacting with CD1d+ cells other

than thymocytes. Multiple CD1d+ cells of hematopoietic, epithelial,

or endothelial origin might influence this process (9).In this study, we sought to gain mechanistic insight into the

functional education of iNKT cells and identify the CD1d-mediated

cellular interactions that regulate their responsiveness.

Materials and MethodsMice

C57BL/6Nmicewere fromCharles River. pLck-hCD1d, mCD1d2/2, pLck-mCD1d, mT, and DDC mice were previously described (8, 10–12).TCRa2/2 mice (13) were obtained from Centre de Distribution, Typage,Archivage Animal–Centre National de la Recherche Scientifique (Orleans,France). Nonviable motheaten (me/+) (C57BL/6J-Hcphme/J) mice werepurchased from The Jackson Laboratories. Mice were kept in a specificpathogen–free environment. All animal studies were reviewed and ap-proved by the Institutional Animal Care and Use Committee, San RaffaeleScientific Institute (protocol N.509).

*Experimental Immunology Unit, Division of Immunology, Transplantation andInfectious Diseases, San Raffaele Scientific Institute, 20132 Milano, Italy;†INSERM U986, Hopital Saint Vincent de Paul/Cochin, Universite Paris Descartes,75014 Paris, France; ‡Department of Infection Biology, University Clinic of Er-langen, Friedrich Alexander University, 91054 Erlangen, Germany; and xLudwig Cen-ter for Cancer Research of the University of Lausanne, 1066 Epalinges, Switzerland

1Current address: Molecular Immunology Unit, Fondazione Istituto di Ricovero eCura a Carattere Scientifico “Istituto Nazionale dei Tumori,” Milan, Italy.

Received for publication December 19, 2012. Accepted for publication January 25,2013.

This work was supported by Associazione Italiana per la Ricerca sul Cancro (11523IG),the Italian Ministry of Health (Ricerca Finalizzata RF-2009-1531842), FondazioneCariplo (Progetto Vaccini 2009-3603), and Regione Lombardia (IMMAGING-SAL-43) to P.D. and G.C.

The sequences presented in this article have been submitted to the Gene ExpressionOmnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42835) under ac-cession number GSE42835.

Address correspondence and reprint requests to Dr. Paolo Dellabona and Dr. GiuliaCasorati, Experimental Immunology Unit, Division of Immunology, Transplantationand Infectious Diseases, San Raffaele Scientific Institute, 20132 Milano, Italy. E-mailaddresses: dellabona.paolo@hsr.it (P.D.) and casorati.giulia@hsr.it (G.C.)

The online version of this article contains supplemental material.

Abbreviations used in this article: BM, bone marrow; DC, dendritic cell; DP, double-positive; iNKT, invariant NKT; iTCR, invariant TCR; LN, lymph node; RFI, relativefluorescence intensity; tg, transgenic; WT, wild-type.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203466

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Flow cytometry

iNKT cells were stained with aGalCer-loaded mouse or human CD1d-IgG1 dimers (BD Pharmingen) (8) or with PBS57-loaded mCD1d-PEtetramers (NIH Tetramer Core Facility), and with mAbs specific forTCRb, CD44, NK1.1, hCD1d, CD3ε, anti-CD45.1, CD24/HSA, CD19,and H-2-Ab (BioLegend). Nonspecific binding was blocked by pre-incubation with anti-CD32/CD16 mAb. Intracellular SHP-1 or cytokineswere detected by staining fixed/permeabilized cells with anti–SHP-1 mAbY476 (Abcam) and secondary Ab (Southern Biotech), or with anti–IFN-gor anti–IL-4 mAbs (BioLegend). The specificity of intracellular SHP-1detection is shown in Supplemental Fig. 2. Samples were acquired onCanto-II flow cytometer (BD Biosciences), excluding aggregates and deadcells, and analyzed using FlowJo (Tree Star). CD69 and SHP-1 expressionlevel was calculated as relative fluorescence intensity (RFI) (sample meanfluorescence intensity/isotype control mean fluorescence intensity).

Cell preparations

Thymocytes, splenic lymphocytes, and hepatic lymphocytes were obtainedas described previously (8). Thymic iNKT cells (TCRbint CD1d-dimers+)and peripheral iNKT cells (CD192MHC-II2 TCRbintCD1d-dimer+ orTCRbintCD1d-tetramer+) were sorted (.95% purity) using a MoFlo cellsorter (Beckman Coulter). Dendritic cells (DCs) were obtained from bonemarrow (BM) cells cultured 5 d with GM-CSF (25 ng/ml) and IL-4 (5 ng/ml; R&D Systems) (14).

iNKT cell activation

For iTCR cross-linking, sorted iNKT cells were placed in 96-well platesprecoated with 10 mg/well goat anti-rat IgG and 2 mg/ml anti-CD28 mAb(BD Pharmingen). Goat anti-rat IgGs cross-links the iTCR by bindingrat anti-mouse IgG1-PE mAbs used to detect the CD1d-dimer+ iNKTcells. For activation by DCs, sorted iNKT cells and DCs were coculturedwith increasing aGalCer concentrations. PMA/ionomycin stimulation wasperformed with 25 ng/ml PMA and 1 mg/ml ionomycin (Sigma) for 1 h at37˚C plus brefeldin A (10 mg/ml) for 1 h. iNKT cells were activated in vivoby injecting 1.25 mg anti-CD3 mAb 3C11 i.v. into mice.

Whole-gene expression analysis by DNA microarray

Total RNA from hepatic iNKT cells was amplified twice, biotin-labeled, andpurified using the RNeasy Mini Kit (Qiagen). The cRNA (10 mg) was hy-bridized to M430 2.0 chips (Affymetrix). All samples were generated intriplicate. Raw data were normalized using the RMA algorithm of Gene-Pattern (15). Nonvariable low expression values were excluded using “Pre-process Dataset” of GenePattern. Data were visualized using the “MultiplotPreprocess,” “Multiplot,” and “Hierarchical Cluster” of GenePattern. Geneontology analysis and visualization were performed using Gorilla software(16). Gene expression data are deposited in National Center for Biotech-nology Information Gene Expression Omnibus and are accessible throughGene Expression Omnibus Series accession number GSE42835 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42835).

Real-time PCR

RNA was amplified using MessageAmp Premier aRNA Amplification(Ambion) and reverse transcribed. TaqMan was performed in triplicate with100 ng cDNA/reaction using specific primers and Gadph or Hprt1 en-dogenous controls (Applied Biosystems) on ABI Prism 7900 and analyzedwith SDS 2.2.1 software. Relative quantitation of gene expression wasdetermined using the comparative cycle threshold method (17). For ab-solute quantification, Ptpn6 exons 8–12 cDNA was cloned into ZeroBluntTOPO Cloning plasmid (Invitrogen). Plasmid DNA was diluted from 109

to 0 copy number into irrelevant DNA. Gapdh and Hprt1 genes wereamplified concurrently to normalize the cDNA of samples. Standard curvesfor both housekeeping genes were prepared using serial dilution of acDNA pool (from 150 to 12.5 ng) derived from wild-type (WT) iNKTcells. Ptpn6 mRNA copy number present in each sample after normaliza-tion was calculated using the standard curves.

BM chimeras

BM chimeras were generated as described previously (11) and analyzed6–8 wk after transplantation.

Statistical analysis

The two-tailed Student t test was used for unpaired samples, applyingcorrection for unequal variances where required. The p values ,0.05 wereconsidered significant.

ResultsiNKT cells from pLck-hCD1d tg mice are hyperresponsive

Functional maturation of iNKT cells requires interaction withCD1d+ APCs, so we investigated reactivity of iNKT cells thatdeveloped in pLck-hCD1d+/2 mCD1d2/2 tg mice (pLck-hCD1dtg) in the absence of “educational” CD1d+ APCs (8). iNKT cellswere sorted from the liver of WT and pLck-hCD1d tg mice usingaGalCer-loaded human CD1d-IgG dimers (hCD1d-dim) and wereactivated by either iTCR cross-linking or aGalCer presentation byDCs. hCD1d-dim bind selectively to mouse Vb8.2+ iNKT cells(18), and the hCD1d transgene selects essentially the same pop-ulation (8), thus enabling robust identification of the same iNKTcell repertoire in both hCD1d tg and WT mice. pLck-hCD1d tg-derived hepatic iNKT cells produced significantly more IFN-g andIL-4 than WT iNKT cells after either iTCR cross-linking (Fig. 1A)or aGalCer-loaded DCs (Fig. 1B). The cells from both mice ex-pressed similar TCR levels (data not shown). To assess whetheriNKT cell hyperreactivity in tg mice depended on enhanced earlyTCR signaling or instead reflected increased capacity for cytokineexpression, we activated hepatic lymphocytes from tg and WT micewith PMA/ionomycin, which induce cytokine production bypassingthe TCR–proximal complex. Similar fractions of iNKT cells fromtg and WT mice expressed CD69 (Fig. 1C, left panels) and pro-duced IFN-g (Fig. 1C, right panels), suggesting that the hyper-reactivity of tg iNKT cells depended on enhanced early TCRsignaling.We next assessed the responsiveness of tg iNKT cells to rapid

activation with anti-CD3 mAb in vivo, because the absence ofCD1d+ APCs in these animals precludes the use of aGalCer.Within 90 min of mAb injection, both splenic and hepatic iNKTcells in tg mice upregulated CD69 and produced significantlymore IFN-g than iNKT cells from WT mice (Fig. 1D). Similarly,IL-4 production was upregulated by tg iNKT cells in liver. In thesesame assays, T cells began to upregulate CD69 similarly in bothanimals, whereas they did not produce cytokines (data not shown).Consistent with the intracellular staining data, tg mice adminis-tered with anti-CD3 mAb exhibited substantially increased IFN-g,and slightly higher IL-4, levels in serum compared with WT mice,upon normalization for the total iNKT cell number present in thespleens of each type of mice (Fig. 1E).Mature T cells of pLck-hCD1d tg mice express low hCD1d levels

(8). However, this leaky hCD1d expression on peripheral T cellplayed a role in the acquisition of NK1.1, but not in the alteredresponsiveness, of iNKT cells from pLck-hCD1d tg mice, as shownby the phenotype and TCR hyperreactivity exhibited by peripheraliNKT cells developing in mixed BM chimeras in which hCD1dexpression was confined to DP thymocytes (Supplemental Fig. 1).Furthermore, the iNKT cell hyperresponsiveness was not due toxenogenic CD1d expression, because iNKT cells isolated from analternative model in which the mouse CD1d cDNA is expressedunder the proximal Lck promoter (10) (pLck-mCD1d tg) exhibitedcomparable Ag hyperreactivity (Fig. 1F). Together, these datasuggested that iNKT cells that develop in pLck-hCD1d tg micedisplay increased responsiveness to TCR-dependent stimulation.

Distinct gene expression profile in iNKT cells frompLck-hCD1d tg mice

To define the molecular basis of iNKT cell hyperreactivity in pLck-hCD1d tg mice, we compared the global gene expression profile ofWTand tg iNKT cells using DNAmicroarrays. Hepatic iNKT cellswere sorted from tg and WT mice using hCD1d-dimers beforeRNAwas extracted for hybridization to genome-wide microarrays(M430v2). iNKT cells fromWTand tg mice exhibited very similar

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transcription profiles, with only 38 known genes upregulatedand 14 downregulated .2-fold in tg cells compared with WTiNKT cells (Fig. 2A, 2B, Supplemental Table I). Gene ontology

analysis of the biological processes associated with these differ-entially expressed genes did not reveal any specific function orpathway (data not shown). The differential expression of a set of

FIGURE 1. iNKT cells from pLck-hCD1d Tg mice are hyperresponsive to TCR-dependent activation. (A) Liver iNKT cells sorted with hCD1d-dimers

from WT or pLck-hCD1d Tg mice and activated by iTCR cross-linking. Cytokines were measured by ELISA after 48 h. Histograms show mean cytokine

concentration 6 SD of one representative experiment with three mice. (B) Sorted iNKT cells were cocultured at 5:1 ratio with DCs loaded with aGalCer.

Cytokines were measured by ELISA after 48 h. (C) Hepatic mononuclear cells (23 105/well) fromWTand pLck-hCD1d tg mice were activated with PMA/

ionomycin for 2 h. CD69 upregulation and intracellular (i.c.) IFN-g production were measured by FACS. Upper panel histograms show one representative

mouse per group; lower panels show mean 6 SD CD69 and IFN-g RFI from the same experiments with four mice per group. (D) CD69 and i.c. cytokine

expression in splenic and hepatic iNKT cells 90 min after i.v. injection of anti-CD3 mAb. Upper panels show staining from one representative mouse per

group. Lower panels show mean 6 SD RFI obtained from the same experiments with four mice per group. (E) Serum cytokines secreted upon anti-CD3

mAb injection as in (D). (F) iNKT cells sorted from pLck-mCD1d tg and control NOD mice were activated with aGalCer-pulsed DCs. Cytokine secretion

was determined by ELISA after 48 h. Cytokine release upon stimulation in vitro is represented as percentage of the cytokines released by WT iNKT cells

activated by either iTCR cross-linking or DCs loaded with the lowest aGalCer concentration. All panels show one representative experiment of three. t test,

*p , 0.05, **p , 0.01, ***p , 0.005.

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these genes with potential roles in the functional regulation ofiNKT cells was validated by RT-quantitative PCR (Fig. 2C).Among the candidate genes downregulated ∼2-fold in tg iNKTcells, Ptpn6 encodes the SHP-1 tyrosine phosphatase that is in-volved in negative regulation of TCR signaling (19, 20). Amongthe confirmed upregulated genes, Ceacam1 encodes Carcinoem-bryonic-Ag–related adhesion molecule 1, which is expressed byactivated T cells and inhibits TCR signaling through interactionwith SHP-1 (21). Also upregulated were genes Tyrobp, encoding

for the NK adaptor protein DAP12, which can exert both acti-vating and inhibitory effects (22), and gene FcgRIIb, encoding thelow-affinity IgG receptor expressed by B cells and activatedT cells that mediates inhibitory functions by recruiting SHP-1,SHP-2, and SH2-containing complex inositol 5-phosphatase SHIP1(23). Finally, upregulated genes Klra5 and Klra7 encode the in-hibitory NK receptors Ly49E and Ly49G2 that recruit SHP-1 viatheir cytoplasmic ITIM regions (24).These data indicated that downregulation of SHP-1 may un-

derpin the increased responsiveness of iNKT cells that develops inthe absence of peripheral CD1d. The upregulation of receptormolecules able to interact with SHP-1 in tg iNKT cells suggestedsome level of functional compensation, as described in NK cellsexpressing inactive SHP-1 (25).

SHP-1 downregulation functionally linked to iNKT cellhyperresponsiveness

To assess a functional link between SHP-1 and iNKT cell reactivity,we first determined that the similarly hyperreactive iNKT cellsfrom pLck-hCD1d and pLck-mCD1d tg mice exhibited also acomparable downregulation of SHP-1 transcript (Fig. 2D). SHP-1transcript was also similarly downregulated in the hyperresponsiveiNKT cells from pLck-hCD1d mice and from BM chimera ex-pressing hCD1d only on DP thymocytes, described earlier (Sup-plemental Fig. 1). Next, we investigated iNKT cell responses inmotheaten mice (me/2), which bear a defective SHP-1 proteinbecause of a single nucleotide deletion in the Ptpn6 gene (26).Mice heterozygous for the motheaten mutation (me/+) exhibit a50% reduction in SHP-1 transcript and protein in hematopoieticcells, resulting in T cell hyperreactivity upon TCR stimulation(26, 27). Unlike the homozygous me/2 mice, the heterozygousme/+ mice are healthy and do not exhibit systemic inflammation(28). Frequency and NK1.1 expression of thymic and hepaticiNKT cells in me/+ and WT mice were comparable (data notshown). We confirmed that both SHP-1 transcript and proteinlevels were similarly reduced in hepatic iNKT cells from bothme/+ and pLck-hCD1d tg mice compared with WT animals (Fig.3A, 3B, Supplemental Fig. 2). As expected, a lower SHP-1 proteinlevel was observed in T cells from me/+ mice, but not in T cellsfrom pLckhCD1d tg mice (Fig. 3B), thereby confirming the se-lective downregulation of SHP-1 in the tg iNKT cells. HepaticiNKT cells sorted from me/+ mice produced more cytokines thancells from controls (me+/+), upon activation either by aGalCer-pulsed DCs (Fig. 3C) or by iTCR cross-linking (Fig. 3D), sup-porting a key role for SHP-1 in regulating iNKT cell respon-siveness.

Restoring contacts with nonthymocyte mCD1d normalizesiNKT cell responsiveness

We next determined whether reintroducing physiological contactwith endogenous mCD1d molecules normalized iNKT cell re-sponsiveness in pLck-hCD1d tg mice. We hence crossed tg andC57BL6 mice to generate pLck-hCD1d tg mCD1d+/+ mice andinvestigated the reactivity of the peripheral iNKT cells. HepaticiNKT cells from pLck-hCD1d tg, pLck-hCD1d tg mCD1d+/+, andWT mice were sorted using hCD1d-dimers and activated in vitroeither by aGalCer-pulsed DCs (Fig. 4A, upper panels) or by iTCRcross-linking (Fig. 4A, lower panels). Both pLck-hCD1d tgmCD1d+/+ and WT iNKT cells produced levels of cytokines sig-nificantly lower than those observed in iNKT cells from pLck-hCD1d tg mice (Fig. 4A). Accordingly, SHP-1 expression wassignificantly increased in both pLck-hCD1d tg mCD1d+/+ and WTiNKT cells compared with pLck-hCD1d tg iNKT cells (Fig. 4B).The hyperreactivity of iNKT cells that develop in pLck-hCD1d tg

FIGURE 2. Whole-gene expression profile of resting iNKT cells from

WT and pLck-hCD1d tg mice. RNAwas extracted from iNKT cells sorted

from the liver of WT and pLck-hCD1d tg mice, and hybridized to

microarrays. (A) Hierarchical clustering of the 77 probes (52 different

genes) differentially expressed in primary WT and tg iNKT cells. Red and

blue indicate relative expression superior or inferior to the gene-wise mean

(GWM). See also Supplemental Table I. (B) Fold change versus expression

plot of primary iNKT cells fromWTand tg mice. Fold change is calculated

as the ratio between MEV of all the probes in tg and WT iNKT cells.

Horizontal lines represent a fold change of 2. Red dots indicate probes that

are significantly higher expressed in tg iNKT cells relative to WT. Green

dots indicate probes with significantly reduced expressed in tg iNKT cells

relative to WT. Differentially expressed molecules with potential roles in

regulating T cell responsiveness are indicated. (C) RT-quantitative PCR on

RNA from sorted hepatic iNKT cells from WT and tg mice. The relative

amount of transcripts in WT and tg iNKT cells was determined by nor-

malization to Gapdh. Results shown are expression fold change (amount of

transcript in tg/amount of transcript in WT iNKT cells). Data are repre-

sentative of three independent experiments. (D) RT-quantitative PCR

performed on RNA extracted from hepatic iNKT cells, sorted from the

indicated tg mice (six mice pooled per strain), using probes specific for

Ptpn6 transcript. Results shown are fold change compared with iNKT cells

from WT mice. One representative experiment of two is shown.

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mice was thus normalized by changes in SHP-1 expression in-duced by contact with CD1d+ cells other than thymocytes.

iNKT cell responsiveness and SHP-1 expression are notimprinted in thymus

To determine whether the iNKT cell hyperreactivity and SHP-1downregulation in pLck-hCD1d tg mice is imprinted in thymus,

iNKT cells from the thymus, spleen, and liver of tg and WT micewere sorted with hCD1d-dimers and then activated by TCR cross-linking to directly compare their effector functions. ThymiciNKT cells from pLck-hCD1d tg and WT mice secreted compa-rable amounts of IFN-g and IL-4 (Fig. 5A), and displayed equiva-lent SHP-1 protein expression (Fig. 5B, Supplemental Fig. 3). InWT animals, peripheral iNKT cells secreted significantly less

FIGURE 3. SHP-1 downregulation and hyperre-

sponsiveness in iNKT cells from motheaten me/+

mice. (A) RNA was extracted from iNKT cells sorted

from the liver of WT, pLck-hCD1d tg, and me/+ mice.

Relative quantification of Ptpn6 transcript by RT-

qPCR (left panel). The amount of Ptpn6 transcript was

normalized to Gapdh, and results are shown as fold

change compared with iNKT cells from WT mice.

Absolute quantification of Ptpn6 transcript by RT-

qPCR (right panel). The number of copies of Ptpn6

mRNA in iNKT cells from WT, pLck-hCD1d tg, and

me/+ mice was determined using a Ptpn6 standard

curve. Samples were normalized to Gapdh RNA. (B)

Upper histograms show intracellular SHP-1 expression

in hepatic T and iNKT cells determined by FACS in

one representative mouse per group. Lower panels

show SHP-1 mean 6 SD RFI in three mice per group.

See also Supplemental Fig. 1. (C) Hepatic iNKT cells

were sorted from the liver of me/+ and littermate WT

mice, and activated either by coculture at 5:1 ratio with

BM-derived aGalCer-loaded DCs, or (D) by iTCR

cross-linking (1 3 104 cell/well). Cytokine production

was measured by ELISA after 48 h. Shown is mean 6SD cytokine production from three mice in one rep-

resentative experiment of three performed. t test, *p ,0.05, **p , 0.01, ***p , 0.005.

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cytokine than thymic iNKT cells. In contrast, cytokine secretion byperipheral iNKT cells from pLck-hCD1d tg was sustained at levelscomparable with those detected in thymic iNKT cells, and cytokineproduction was significantly higher than that displayed by WTperipheral iNKT cells. The expression of SHP-1 protein increasedmarkedly from thymus to periphery in iNKT cells from WT mice,but not in iNKT cells from pLck-hCD1d tg mice (Fig. 5B). Thus,under physiological conditions, iNKT cells upregulate SHP-1 ex-pression in the peripheral compartment. Both the NK1.12 andNK1.1+ subsets of peripheral iNKT cells from pLck-hCD1d tgmice displayed similar reductions in SHP-1 protein expressioncompared with the corresponding populations in WT animals (Fig.5C), indicating that reduced SHP-1 expression by tg iNKT cells isindependent of their maturation.Together, these data suggested that iNKT cell responsiveness and

SHP-1 expression in pLck-hCD1d tg mice are not imprinted inthymus. Instead, tg iNKT cells fail to upregulate SHP-1 expressionin the absence of CD1d contacts in the periphery.

Functional education of iNKT cells is mediated by CD1d+ DCs

To identify the CD1d+ cells that might regulate iNKT cell re-sponsiveness in peripheral compartments, we considered B cellsand DCs as primary candidates, because both cell types expressCD1d and interact with iNKT lymphocytes (29). Furthermore,hyperreactivity of peripheral iNKT cells from pLck-mCD1d tg

mice has been reported to normalize upon crossing these animalswith tg mice that express mCD1d on MHC II+ APCs (7).We first investigated the reactivity of iNKT cells in mT mice that

lack B lymphocytes (30). The percentage of iNKT cells in theseanimals was normal in thymus and in periphery, but the numberswere decreased in periphery (data not shown), consistent with theobserved T and NK cell reduction in these animals (31, 32).Splenic and hepatic iNKT cells of mT and WT mice that weresorted and activated in vitro by TCR cross-linking were compa-rably responsive, although IL-4 secretion by splenic iNKT cellswas somewhat reduced in mT mice (Fig. 6A). The normal re-sponsiveness of peripheral iNKT cells from mT mice was asso-ciated with normal levels of SHP-1 protein expression (Fig. 6B).The role of DCs in the regulation of iNKT cell responsiveness

was investigated using mice in which DCs are constitutively de-leted by the expression of an inducible tg diphtheria toxin (DDCmice) (12). DDC mice display .90% reduction in myeloid DCs,lymphoid DCs, plasmacytoid DCs, and Langerhans cells in thy-mus, spleen, lymph nodes (LNs), and skin, leading to lethal sys-temic autoimmunity by 7–8 wk of age (12). We therefore sortedliver iNKT cells from 5-wk-old DDC mice and WT mice usingmCD1d dimers, and activated these cells by iTCR cross-linkingin vitro. iNKT cells from DDC mice were hyperresponsive (Fig.6C), suggesting that DCs are key regulators of the iNKT cellactivation threshold. To confirm these data in mice not affected

FIGURE 4. Coexpression of mCD1d and hCD1d

normalizes iNKT cell responsiveness and SHP-1 ex-

pression. (A) Hepatic iNKT cells were sorted from

pLck-hCD1 tg, pLck-hCD1d mCD1d+/+, and WT

mice. iNKT cells (2 3 104/well) were activated by

aGalCer-loaded WT DCs (4 3 103/well; upper panels)

or by iTCR cross-linking (104 cells/well; lower panels).

Cytokines were measured by ELISA after 48 h. Values

significantly different between WT and pLck-hCD1d

iNKT cells (asterisk); values significantly different

between pLck-hCD1d mCD1d+/+ and pLck-hCD1d

iNKT cells (boxed asterisk). Shown are mean 6 SD of

cytokine produced by three mice per group from one

representative experiment of three. (B) SHP-1 intra-

cellular (i.c.) staining of T and iNKT cells from the

liver of the indicated mice. Upper panels show SHP-1

i.c. expression from one representative mouse per

group. Lower panels show SHP-1 mean6 SD RFI from

all three mice per group. One representative experiment

of three is shown. t test, *p , 0.05, **p , 0.01.

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by autoimmune pathology, we reconstituted lethally irradiatedCD1d2/2 mice with 20% BM cells from either WT mice orCD1d2/2 mice in combination with 80% BM cells from DDCmice (Fig. 6D). The frequencies of iNKT cells were similar in thetwo types of BM chimera (Fig. 6E), but the proportion of matureNK1.1+ iNKT cells in the liver and spleen was reduced in animalsthat lacked CD1d+ DCs (Fig. 6E). iNKT cells from mice thatlacked CD1d+ DCs were also hyperresponsive to activation invitro (Fig. 6F). Consistent with this hyperreactivity, iNKT cellsfrom chimera deficient in CD1d+ DCs expressed a lower SHP-1mRNA copy number compared with cells from control chimera(Fig. 6G). In the thymus, iNKT cells from BM chimera deficientin CD1d+ DCs displayed normal reactivity, and their SHP-1transcript copy number was comparable with that expressed byWT thymic iNKT cells (data not shown).Collectively, these data indicated that stimulation by CD1d

expressed on peripheral DCs functionally educates iNKT cells bytuning SHP-1 expression.

DiscussionOur study reveals a novel mechanism of iNKT cell functionaleducation that occurs in the periphery and is exerted by tuning the

TCR signaling threshold. This process is mediated by the upreg-ulation of SHP-1 in iNKT cells, induced upon engaging CD1d+

DCs. In the absence of this functional checkpoint, expression ofSHP-1 remains low, resulting in altered reactivity of iNKT cells.Tyrosine phosphatase SHP-1 exerts a global negative regulatory

function on TCR-mediated activation of T lymphocytes by dephos-phorylating several proximal signal transduction mediators, therebysetting the threshold for thymocyte selection and activation ofmature T cells (20). SHP-1 levels are physiologically modulatedduring T cell development or upon activation, increasing theavidity of immature thymocytes and recently activated T cells forAg signals (20). Our finding that iNKT cells upregulate SHP-1 andreduce TCR responsiveness in the periphery is consistent withrecent data from tg mice expressing a GFP reporter that directlyresponds to TCR signaling (33). Thymic iNKT cells from theseanimals are brightly fluorescent, whereas peripheral iNKT cellsare not, suggesting that these cells may become desensitized toCD1d stimulation in the periphery.DCs play a key role in the activation of iNKT cells (29) and

mediate their thymic negative selection (8, 34). B cells are abun-dant CD1d+ APCs in secondary lymphoid organs and are also ableto interact with iNKT cells (35–37). Yet, our data suggest that DCs

FIGURE 5. iNKT cell hyperreactivity in pLck-hCD1d tg mice is not imprinted in thymus. (A) iNKT cells were sorted from the thymus, spleen, and liver

of WT or pLck-hCD1d tg mice and activated by iTCR cross-linking. Cytokines were measured by ELISA after 48 h. Shown are mean 6 SD cytokine

produced by iNKT cells sorted from six mice per group. Cytokine release is represented as percentage of the cytokines released by WT hepatic iNKT cells.

(B) SHP-1 intracellular staining of iNKT cells from WT and pLck-hCD1d tg thymus, spleen, and liver. Shown is the mean 6 SD SHP-1 RFI obtained from

one representative experiment of three performed with four mice per group. (C) Mean6 SD SHP-1 RFI in NK1.12 and NK1.1+ iNKT cells from spleen and

liver of WT and pLck-hCD1d tg mice. See also Supplemental Fig. 2. One representative experiment of two performed with four mice per group is shown.

t test, *p , 0.05, **p , 0.01, *** p , 0.005.

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are key in tuning the TCR responsiveness of iNKT cells in pe-ripheral tissues in the steady-state. DCs, unlike B cells, mightprovide costimulatory signals that, in addition to the CD1d-de-pendent one, may cooperate to upregulate SHP-1 expression byperipheral iNKT cells. iNKT cells might also preferentially con-tact DCs in unchallenged situations, as they colocalize with DCsin the spleen marginal zone and periarteriolar lymphoid sheaths,LNs subcapsular area, and liver sinusoids (38–40). We cannotexclude that also CD1d+ macrophages, which locate proximally toiNKT cells in the splenic marginal zone and in LNs (38, 40), andmay be depleted in DDC mice, may contribute to tuning iNKT cellresponsiveness. Conditional deletion of CD1d expression by DCswas recently reported to exert little effect on iNKT responsiveness(41). This model is different from ours, and a differential persis-tence of residual CD1d+ DCs in these mice and in the DDC miceused in this study might account for the variable responsivenessobserved.We show that B lymphocytes do not play a major role in reg-

ulating SHP-1 expression in iNKT cells. However, upon TCRcross-linking in vitro, we find a reduced IL-4 secretion by splenic

iNKT cells from mT mice. Upon aGalCer injection in vivo,a normal iNKT cell response was found either in mT mice (42) orin mice that exhibit only CD1d-deficient B cells (41), whereasother studies suggested a possible CD1d-dependent suppressiverole for B cells in regulating iNKT cell function (29). It is difficultto compare the different iNKT cell activation regimens used inthese studies; nevertheless, these data imply that B cells mightalso regulate iNKT cell effector response, possibly in specificanatomical sites and likely by mechanisms distinct from DCs.Peripheral contacts with CD1d are required for the final phe-

notypic maturation of iNKT cells (7, 43, 44). We find that contactswith peripheral CD1d+ DCs results in the upregulation of bothNK1.1 and SHP-1 expression by iNKT cells. iNKT cells maycomplete their maturation upon engaging self-lipid Ags (45), aswell as exogenous lipid(s) derived from the intestinal microbiotaof mice (46), following uptake and presentation by CD1d-expressingDCs. We also show that hCD1d expression by peripheral T cellsin the pLck-hCD1d tg mice can upregulate NK1.1, but not SHP-1,in iNKT cells. Human CD1d may deliver a stronger “basic” stimu-lation to peripheral iNKT cells than mouse CD1d (8), sufficient

FIGURE 6. CD1d+ DCs functionally

educate iNKT cells. (A) Splenic and

hepatic iNKT cells from WT and mT

mice (1.5 3 104 cells/well) were acti-

vated by iTCR cross-linking. Secreted

cytokines were measured by ELISA af-

ter 48 h. Mean 6 SD cytokine produc-

tion from five mice per group is shown.

(B) SHP-1 intracellular staining by

FACS in T and iNKT cells from spleen

and liver from WT and mT mice. Upper

panels are from one representative

mouse per group. Lower panels show

mean 6 SD SHP-1 RFI obtained from

four mice per group. One representative

experiment of two is shown. t test, *p ,0.05 **p, 0.01. (C) Hepatic iNKT cells

from WT and DDC mice (1.5 3 104

cells/well) were activated by iTCR

cross-linking. Cytokines were measured

by ELISA after 48 h. Mean 6 SD cy-

tokine production from four mice per

group is shown. (D) PBMCs from BM

chimeras were analyzed for CD1d+ DCs.

Shown is one representative BM chi-

meric mouse (CD1d, black line; isotype

control, gray filled histogram). (E) iNKT

cell frequency and NK1.1 expression in

thymus, spleen, and liver of chimeric

mice. Shown is the percentage of gated

population from five mice pooled per

chimera. (F) iNKT cells from spleen and

liver from the BM chimeras (1.5 3 104

cells/well) were activated by iTCR

cross-linking. Cytokines were measured

by ELISA after 48 h. Shown are mean6SD cytokine produced by iNKT cells

from five mice per group. (G) Absolute

Ptpn6 mRNA quantification in iNKT cells

sorted from the spleen and liver of chi-

meric mice. Samples were normalized to

Gapdh, and Ptpn6 gene copy number was

measured using a Ptpn6 standard curve.

Shown is one representative experiment of

two. t test, *p , 0.05, **p , 0.01.

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for NK1.1 upregulation. SHP-1 upregulation in peripheral iNKTcells may require additional costimulatory signals, selectively pro-vided by DCs and not by B or T lymphocytes.Unlike their peripheral counterparts, thymic iNKT cells from

pLck-hCD1d tg mice exhibited normal reactivity and SHP-1 levels,in line with published data showing that thymic and peripheralNKT cells represent different functional subsets (7, 47), controlledby distinct molecular and/or environmental cues (7, 48).The mechanisms by which CD1d+ DC-dependent signaling up-

regulates SHP-1 expression in peripheral iNKT cells remain to bedefined. In a different context, it was recently shown that weakstimulation of human iNKT cells by CD1d-expressing APCsin vitro opens the IFNG locus and makes it accessible to tran-scription triggered by innate signaling (49). The TCR triggering ofmouse iNKT cells by CD1d+ DCs may poise the PTPN6 locus totranscription, induced by concomitant costimulatory signals di-rectly delivered by DCs or present in the microenvironment.In conclusion, our study defines a functional education step of

iNKT cells occurring in the periphery, which may be critical tocontrol the innate effector state of iNKT cells by tuning their re-activity toward stronger stimulatory signals, generated in stress-related conditions and bacterial infections.

AcknowledgmentsWe thank L. Guidotti and C. Benoist for critical manuscript reading.

DisclosuresThe authors have no financial conflicts of interest.

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