of April 13, 2018.This information is current as

CD1d-Restricted Mouse NKT CellsProducing−Development of IL-17

ZBTB7B (Th-POK) Regulates the

GodfreyKonstantinos Kyparissoudis, Chris C. Goodnow and Dale I. Sandra Frankenreiter, Hannes Bergmann, Carla M. Roots,Uldrich, Mehmet Yabas, Torsten Juelich, John A. Altin, Anselm Enders, Sanda Stankovic, Charis Teh, Adam P.

http://www.jimmunol.org/content/189/11/5240doi: 10.4049/jimmunol.1201486October 2012;

2012; 189:5240-5249; Prepublished online 26J Immunol 

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6.DC1http://www.jimmunol.org/content/suppl/2012/10/26/jimmunol.120148

Referenceshttp://www.jimmunol.org/content/189/11/5240.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

ZBTB7B (Th-POK) Regulates the Development ofIL-17–Producing CD1d-Restricted Mouse NKT Cells

Anselm Enders,* Sanda Stankovic,† Charis Teh,* Adam P. Uldrich,† Mehmet Yabas,*

Torsten Juelich,‡ John A. Altin,‡ Sandra Frankenreiter,* Hannes Bergmann,*

Carla M. Roots,‡,x Konstantinos Kyparissoudis,† Chris C. Goodnow,‡,x,1 andDale I. Godfrey†,1

CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4+CD82 and CD42CD82 subsets that

respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination

of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze

a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation

has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant

mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant

NKT cells in the thymus are retinoic acid–related orphan receptor gt positive, and a high frequency produce IL-17 while very few

produce IFN-g or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation

also have reduced numbers of CD4+ NKT cells and increased production of IL-17 without an increase in CD8+ cells, suggesting that

ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermin-

ing the balance of effector subsets within the NKT cell population. The Journal of Immunology, 2012, 189: 5240–5249.

The factors that regulate formation of distinct subsetsof effector T cells are not well understood. While theseresponses are clearly influenced by the nature and route of

exposure of an encountered Ag, genetic wiring also influences thekinds of effector T cell responses. Understanding these genetic

factors is important to explain individual variability in physio-logical or pathological immune reactions to common Ags.NKT cells are CD1d-restricted, glycolipid Ag-reactive T cells

that represent a unique population of effector T cells in miceand humans. These cells express a heavily biased TCR repertoire,composed of an invariant TCR a-chain (Va14Ja18 in mice,Va24Ja18 in humans) paired with a limited array of TCRb-chains (Vb8.2, Vb7, or Vb2 in mice, Vb11 in humans) (1, 2).NKT cells can influence a broad spectrum of diseases, rangingfrom suppression of autoimmune diseases like type 1 diabetes, topromotion of immunity, to cancer and infection (3). This paradox-ical ability to promote or suppress immune responses is associatedwith the profound ability of NKT cells to produce a spectrum ofcytokines within hours of stimulation. At the population level,NKT cells produce seemingly antagonistic cytokines including IFN-g, IL-4, IL-10, IL-13, and IL-17, although NKT cells can be dividedinto functionally distinct subsets that are capable of preferentiallyproducing only some of these cytokines (4–7), which may partlyexplain the diverse functional outcomes associated with these cells.Human NKT cells vary widely in frequency between individuals,

yet are stable within individuals (8, 9). Human NKT cells includeCD4+, CD42CD82 (double negative [DN]), and CD8+ subsets, andeach of these exhibit distinct cytokine profiles, which again suggeststhat they have distinct functions in vivo. The ratio of CD4/CD8 de-fined subsets of NKT cells also varies widely between individuals(10). Given this variability, combined with their powerful immuno-regulatory potential, it is very important to decipher the factors thatregulate NKT cell development and homeostasis, including factorsthat determine the balance of functionally distinct NKT cell subsets.Many molecules, including cell surface receptors, signal trans-

duction and transcription factors, have been identified that selec-tively regulate NKT cell numbers independently from conventionalT cells (11). For example, the SLAM/SAP/fyn signaling pathwayis selectively important for NKT cell development while dispens-able for T cell development in the thymus (11). However, little is

*Ramaciotti Immunization Genomics Laboratory, Department of Immunology, JohnCurtin School of Medical Research, Australian National University, Canberra, Aus-tralian Capital Territory 0200, Australia; †Department of Microbiology and Immu-nology, University of Melbourne, Parkville, Victoria 3010, Australia; ‡Department ofImmunology, John Curtin School of Medical Research, Australian National Univer-sity, Canberra, Australian Capital Territory 0200, Australia; and xAustralian Phenom-ics Facility, Australian National University, Canberra, Australian Capital Territory0200, Australia

1D.I.G. and C.C.G. contributed equally to this work.

Received for publication May 29, 2012. Accepted for publication September 26,2012.

This work was supported by research grants from the Wellcome Trust, the NationalInstitutes of Health (AI054523), the National Health and Medical Research Councilof Australia (NHMRC), the CASS Foundation, and the Ramaciotti Foundations. A.E.was supported by a Deutsche Forschungsgemeinschaft Research Fellowship (EN790/1-1) and an NHMRC Project Grant (APP1009190) and Career DevelopmentFellowship (APP1035858). D.I.G. was supported by an NHMRC Senior PrincipalResearch Fellowship (1020770). C.C.G. was supported by an NHMRC AustraliaFellowship (585490) and by an Australian Research Council Federation Fellowship.

A.E., C.C.G., and D.I.G. planned the experiments and wrote the article, and A.E.,S.S., C.T., A.P.U., T.J., H.B., J.A.A., S.F., M.Y., C.M.R., and K.K. performed theexperiments.

The funders of the study had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Address correspondence and reprint requests to Prof. Dale I. Godfrey or Prof. ChrisC. Goodnow, Department of Microbiology and Immunology, University of Mel-bourne, Parkville, VIC 3010, Australia (D.I.G.) or Department of Immunology, JohnCurtin School of Medical Research, Australian National University, Canberra, ACT0200, Australia (C.C.G.). E-mail addresses: godfrey@unimelb.edu.au (D.I.G.) andchris.goodnow@anu.edu.au (C.C.G.)

The online version of this article contains supplemental material.

Abbreviations used in this article: DN, double negative; LN, lymph node; RORgt,retinoic acid–related orphan receptor gt; WT, wild-type.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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known about what regulates the differentiation of NKT cell sub-sets. One study, an investigation of NKT cell development inGATA-3 knockout mice, provided data showing that CD4+

NKT cells were preferentially inhibited in the absence of thisfactor (12). What factors regulate the appropriate expression of theCD4 or CD8 coreceptors in MHC class II– or MHC class I–re-stricted thymocytes has itself been a long-standing issue. In 2005,two studies showed that the transcription factor ZBTB7B (previ-ously called Th-POK and cKrox) plays a key role in maintainingCD4 expression in MHC class II–restricted thymocytes (13, 14).ZBTB7B promotes CD4 expression indirectly by preventingRunx1- and Runx3-mediated downregulation of CD4 in conven-tional TCRab T cells (15). It has been shown subsequently thatCD4 expression by NKT cells is also dependent on ZBTB7B (16,17). Furthermore, in the absence of ZBTB7B, NKT cell cytokineproduction was impaired, which led to the suggestion that ZBTB7Bis required for full NKT cell maturation and activation (16).In this study, we describe a novel mouse strain, termed “help-

less,” carrying a point mutation in Zbtb7b creating a single aminoacid substitution in the BTB-POZ domain. Using this mousemodel, we show that ZBTB7B plays an essential, cell intrinsic,and dose-dependent role in establishing the balance of differentNKT cell subsets, including maintenance of CD4, inhibition ofCD8 expression, and development of the retinoic acid–relatedorphan receptor gt (RORgt)-positive IL-17+ population of NKT cells.ZBTB7B thereby establishes a genetically predetermined profile ofCD1d-restricted NKT cells.

Materials and MethodsMice

The Zbtb7bhpls/hpls strain derived from a C57BL/6 male treated three timesi.p. with 100 mg/kg N-ethyl-N-nitrosourea at weekly intervals. Mice weremaintained on a pure C57BL/6 background or on a mixed CBA 3 C57BL/6background. All mice were housed in specific pathogen–free conditions atthe Australian Phenomics Facility. All experimental procedures were ap-proved by the Australian National University Animal Ethics and Experi-mentation Committee.

Sequencing and genotyping

All exons and splice sites of Zbtb7b were amplified, and primers for se-quencing were designed using Australian Phenomics Facility software toamplify all exons and splice sites for Zbtb7b. The amplification and dualsequence run were performed at the Brisbane node of the Australian GenomeResearch Facility. Sequence analysis was conducted at the Australian Phe-nomics Facility using Lasergene software (DNAStar). A T to G substitu-tion of bp 480 was identified in exon 2 (ensmuse000001765423) of Zbtb7b,resulting in a CTG (Leu) to a CGG (Arg) amino acid change. Mice weregenotyped using an Amplifluor assay (Chemicon). All primer sequencesare available on request.

Flow cytometry

Cell suspensions from thymus and spleen were prepared by passing the cellsthrough a cell strainer (BD Biosciences) or stainless steel sieve, followedby lysis of RBCs for spleen and liver samples. Liver lymphocytes wereisolated by centrifugation over a Percoll gradient. Cell suspensions werelabeled with fluorochrome-coupled Abs according to standard protocolsand run on an LSR II or Canto flow cytometer (BD Biosciences) followedby analysis with FlowJo (Tree Star). a-GalCer–loaded CD1d tetramers wereproduced in house, using a mouse CD1d baculovirus construct originallyprovided by Prof. Mitchell Kronenberg, as previously described (18). Forsome experiments, a-GalCer (PBS57)-loaded CD1d tetramers provided bythe National Institutes of Health Tetramer Facility were used.

In vitro stimulation and cytokine analysis

For the intracellular cytokine staining assay, cells were stimulated with 50ng/ml phorbol ester and 500 ng/ml ionomycin for 2.5–3.5 h at 37˚C in thepresence of monensin in RPMI 1640 culture media supplemented with10% heat-inactivated FCS, glutamine (Life Technologies), 10 mM sodiumpyruvate (Life Technologies), 10 mM HEPES (Life Technologies), 10 mM

MEM nonessential amino acids (Life Technologies), and 5.5 mM 2-mer-captoethanol. Stimulated cells were then washed, stained for surfacemarkers, and stained intracellularly using FITC-conjugated anti-mouseIL-17A (BioLegend) or isotype-matched control Abs (BD Pharmingen),RORgt–PE, or allophycocyanin (eBioscience) using the eBiosciencefixation/permeabilization kit.

For cytometric bead array, thymocytes were pooled from several mice pergroup and enriched by either staining with anti-CD24 (J11D) followed bydepletion using rabbit complement (C-SIX Diagnostics) in the presenceof DNase (Roche Diagnostics) or by staining cells with PE-conjugatedCD1d–a-GalCer tetramer and subsequent incubation with anti-PEmicrobeads (Miltenyi Biotech). Labeled cells were then enriched bypassing them through a magnetic column and were further stained for flowcytometric purification. This second method was also used to enrichsplenic dendritic cells (based on CD11c expression). Enriched cells weresorted using a FACSAria (BD Biosciences) in the Department of Micro-biology and Immunology Flow Cytometry Facility (University of Mel-bourne) to obtain highly purified populations. Sorted NKT cells werestimulated by placing them in 96-well plates (2 3 104 cells/well), coatedwith anti-CD3 and anti-CD28, or with soluble CD1d loaded witha-GalCer, or by coculture with sorted splenic dendritic cells (1 3 104

dendritic cells/well) loaded with a-GalCer. After 24 h, the supernatant wascollected and the concentration of cytokines secreted into the mediumdetermined by cytometric bead array (BD Pharmingen).

Cell sorting, RNA extraction, cDNA synthesis, and quantitativePCR

Single-cell suspensions from thymocytes were prepared and stained as forflow cytometric analysis. Samples were sorted on a FACSAria sorter (BDBiosciences) at the Flow Cytometry Facility of the John Curtin Schoolof Medical Research (Australian National University). Total RNA wasextracted using RNA TRIzol reagent (Molecular Research Centre) andreverse transcribed using random oligonucleotide primers and 50 USuperscript II reverse transcriptase (Invitrogen) as detailed in themanufacturer’s guidelines. SYBR Green real-time PCR reactions wereperformed in 96-well plates (PerkinElmer) with an ABI PRISM 7900 Real-Time System (PerkinElmer/PE Biosystems) at the Biomolecular ResourceFacility (John Curtin School of Medical Research, Australian NationalUniversity). To correlate the threshold (Ct) values from the cDNA am-plification plots to fold differences between samples, the DDCt method wasapplied using the housekeeping gene GAPDH.

Generation of bone marrow chimeras

B6.SJL CD45.1 mice were irradiated with 10 Gy and injected with 23 106

bone marrow cells consisting of a 50:50 mix of wild-type (WT) B6.SJLCD45.1+ and either WT or mutant C57BL/6 (CD45.2+) cells. They wereallowed to reconstitute for 8 wk before analysis.

ResultsIdentification of the Zbtb7b mutant strain helpless (hpls)

In a genome-wide screen for N-ethyl-N-nitrosurea–induced pointmutations affecting the development of the immune system (19),we identified a strain that was deficient in CD4+ T cells in pe-ripheral blood and spleen. Further analysis revealed a block inCD4 development at the CD4+CD8dim stage in the thymus (Fig.1A). This phenotype is identical to the phenotype described for theHelperDeficient (HD) strain caused by an amino acid substitutionin the DNA-binding zinc finger domain of the transcription factorZBTB7B, previously called Th-POK or cKrox (13, 20) orknockout mice with a Zbtb7b null allele (21). Because of thesesimilarities, we sequenced Zbtb7b and identified a mutationchanging a conserved leucine to arginine in the BTB-POZ domain(Fig. 1B). BTB-POZ domains mediate homodimerization andheterodimerization, association with nuclear corepressors, andubiquitination (22). Deletion of the BTB-POZ domain from aZbtb7b transgene inactivated its ability to deviate MHC class I–restricted T cells into the CD4 lineage (14). The leucine that ismutated in helpless mice is buried within the homologous PZLFBTB-POZ domain (Supplemental Fig. 1 and Ref. 23). TheZBTB7BL102R disrupts CD4 cell differentiation as completely asthe null mutation, but whether this reflects misfolding of the BTB

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domain or destabilization of the ZBTB7B protein as a whole orloss of particular protein–protein interactions is unclear. Geno-typing of affected and unaffected progeny from numerous helplesscarriers confirmed that the failure of CD4 cell differentiation wasinherited in complete concordance with the Zbtb7b mutation in arecessive fashion. As noted previously for the HD strain (24), ho-mozygous affected mice on the parental C57BL/6 background wereborn at around half the expected frequency, and affected miceshowed poor breeding efficiency. This was rescued by keeping themice on a mixed C57BL/63 CBA background, where homozygoteswere obtained at Mendelian ratios. The embryonic lethality onthe C57BL/6 background may indicate essential functions forZBTB7B in other processes such as collagen gene regulation (25).It was previously reported that CD4 expression by NKT cells is

disrupted in Th-POK–deficient mice (16, 17), so we first investi-gated whether the Zbtb7b point mutation in hpls/hpls mice hada similar impact on these cells. Normally, around 70% of spleen

NKT cells express CD4, and none are CD8+. In the helpless strain,this was completely reversed with ∼70–80% of NKT cells in thespleen expressing CD8, and none were CD4+ (Fig. 1C).Examination of NKT cells in different tissues revealed that the

percentage in thymus, spleen, and liver was comparable betweenWT and hpls/hpls mice, whereas the percentages and absolute cellnumbers of these cells in blood, bone marrow, and lymph nodes(LNs) were clearly higher (Fig. 1D, 1E). Notably, Zbtb7b het-erozygous mice also showed an increased percentage of NKT cellsin thymus, LN, and bone marrow, but not in spleen and liver (Fig.1D). The CD8+ NKT cell phenotype was detectable in thymus ofhpls/hpls mice (∼20% CD8+) but far more pronounced in thespleen and liver where ∼80% were CD8+ (Fig. 1F). Further analysisof the CD8+ NKT cells in thymus, spleen, and liver showed thatthey included some cells that were CD8a+b2 and some thatwere CD8a+b+ (Fig. 1G). Furthermore, heterozygous hpls/+mice exhibited an intermediate phenotype, where CD4+ NKT

FIGURE 1. CD8+ NKT cells predominate in a Zbtb7b mutant mouse strain. (A) Homozygotes for the helpless mutation (hpls/hpls) have reduced

percentage of CD4+ T cells in the peripheral blood and spleen and a block in thymic T cell development at the CD4+CD8dim stage. (B) T480G mutation in

Zbtb7b exon 1, altering codon 102 from leucine to arginine within the BTB-POZ domain. The bottom panel shows an alignment of parts of the BTB-POZ

domains from the indicated proteins, with the mutated residue highlighted in red. (C) Flow cytometric analysis of spleen cells stained with a-GalCer–loaded

CD1d tetramers and Abs to TCR, CD4, and CD8. Numbers in the upper panels show percentage of spleen cells within the gate, and lower panels show the

percentage of these gated NKT cells that are CD4+, CD8+, or negative for both. (D) Percentage of NKT cells in thymus, blood, spleen, LN, bone marrow

(left axis), and liver (right axis) of Zbtb7bhpls/hpls, Zbtb7bhpls/+, and Zbtb7b+/+ mice. Each data point represents a different mouse, and the bars represent the

mean. The data for the percentages and absolute cell numbers of NKT cells in thymus, spleen, and liver are pooled from at least two (liver and bone

marrow) or more different experiments with some animals for the liver data on a mixed CBA 3 C57BL/6 background. For liver, mixed background data

points are depicted with hexagons, pure B6 data points are depicted with triangles. (E) Absolute cell number of NKT cells in thymus, spleen, bone marrow

(left axis) and LN and liver (right axis) of Zbtb7bhpls/hpls, Zbtb7bhpls/+, and Zbtb7b+/+ mice. Each data point represents a different mouse, and the bars

represent the mean. (F) Percentage of CD4+, CD82; CD42, CD82; and CD42, CD8+ NKT cells within the thymus, spleen and liver of Zbtb7b1/1, Zbtb7bhpls/1

and Zbtb7bhpls/hpls mice. Each data point represents a different mouse. (G) Expression of CD8 a-chain and CD8 b-chain on CD42 NKT cells from WT and

mutant mice in thymus, spleen, and liver. Except for liver data for hpls/+ mice, all flow cytometric data are representative of at least three different experiments

with at least two animals per genotype and experiment. Statistics were calculated using the Kruskal–Wallis test. *p , 0.05, **p , 0.005, ***p , 0.0005.

5242 DEVELOPMENT OF IL-17 PRODUCING NKT-CELLS DEPENDS ON ZBTB7B

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cells were reduced, yet there was no sign of CD8 upregulation, inboth thymus and periphery, thus hpls/+ mice were highly enrichedfor CD42CD82 NKT cells (Fig. 1F). This contrasts with the de-velopment of conventional CD4+ T cells that is seemingly unaf-fected in heterozygous mice (Fig. 1A) strongly suggesting that theeffect on NKT surface marker expression is not due to a dominant-negative effect of the hpls mutation.

NKT cell development is abnormal in Zbtb7b mutant mice

The developmental events giving rise to the unusual CD8+

NKT cell phenotype in Zbtb7b mutant mice have not been de-termined; however, the accumulation of these cells in peripheraltissues at much higher levels than in thymus suggested this occursas a late event in NKT cell development. Therefore, we morecarefully examined NKT cells in the thymus to determine theorigins of this defect. Analysis of Zbtb7b mRNA expression byreal-time PCR revealed that both immature NK1.12 and matureNK1.1+ NKT cells expressed Zbtb7b (data not shown). Zbtb7bexpression levels were similar to CD4+CD8dim thymocytes, lowerthan total CD4+ single-positive thymocytes, but clearly abovedouble-positive and CD8 single-positive thymocytes that haveexpression at or just above background (13). Because NKT cellnumbers in the thymus of Zbtb7b mutant mice were comparable to

those in WT mice, this suggested there was no major problem withthe selection and expansion of these cells as a total population.NKT cell development can be divided into three developmentallydistinct stages: stage 1 (CD44loNK1.12); stage 2 (CD44+NK1.12);stage 3 (CD44+NK1.1+) (26, 27); and these stages can be furtherdivided into CD4+ and CD42, a split that occurs at approximatelystage 2 (4). Although hpls/hpls NKT cells were mostly mature, asdefined by their NK1.1+CD44hi phenotype, they expressed slightlylower levels of NK1.1 (Fig. 2A). Although CD8+ NKT cells weredetected in the hpls/hpls thymus, the high-intensity CD8 expres-sion observed with hpls/hpls peripheral NKT cells (Fig. 1C) wasnot reflected in the thymus where they were mostly CD8lo. Thissuggested that the emergence of CD8+ NKT cells begins in thethymus but is not fully manifested until these cells are in theperiphery. Examination of increasingly mature NKT cell subsetsrevealed that low CD8 expression was detectable from the earliestCD44loNK1.12 stage, but the percentage of CD8+ cells increasedas the NKT cells matured through CD44+NK1.12 and CD44+

NK1.1+ stages (Fig. 2A, 2B).To determine whether the defects in NKT cell development in

hpls/hpls mice were cell intrinsic, we performed mixed bone mar-row chimera experiments to compare WT and hpls/hpls NKT cellsdeveloping in the same environment. These results demonstrated

FIGURE 2. Divergent NKT cell development in the

thymus of Zbtb7b mutant mice. (A) Thymic NKT cells,

gated on a-GalCer–CD1d tetramer+ and TCRb+ cells,

showing subsets resolved by CD44 and NK1.1 expres-

sion. The bottom panels are further gated on CD44+

NK1.1+ mature NKT cells, showing CD4 and CD8 ex-

pression. (B) The percentage of CD8+ NKT cells within

each stage of hpls/hpls NKT cell maturation in the thy-

mus, as shown in (A) (second row). Each symbol rep-

resents an individual mouse; data are from at least four

independent experiments with two to four mice per

group. (C) Mixed bone marrow chimera results showing

relative percentage of CD4/CD8 defined NKT cell sub-

sets in thymus. Each symbol represents a different re-

cipient mouse in a single experiment. Equal numbers of

CD45.1+ +/+ and CD45.2+ hpls/hpls bone marrow cells

were used to reconstitute irradiated CD45.1 recipients.

After hemopoietic reconstitution, thymocytes of indi-

vidual animals were analyzed by flow cytometry to

identify NKT cell subsets from either WT or hpls bone

marrow origin in the same animal. (D) Relative contri-

bution of hpls/hpls and WT cells to the stages of NKT

cell maturation in mixed bone marrow chimeras, gener-

ated as described in (C) (filled circles) and control chi-

meras generated from an equal mix of CD45.1+ +/+ and

CD45.2+ +/+ bone marrow cells (open circles). To ac-

count for small interindividual differences in overall

hemopoietic reconstitution, the CD45.2/CD45.1 cell ra-

tios in NKT cell subsets of each animal were normalized

by dividing by the ratio in DP thymocytes in the same

mouse. (E) Percentage of NKT cells staining for the cell

cycle marker Ki67 in individual mixed bone marrow

chimeras, gated on CD45.2+ hpls/hpls cells (filled circles)

or CD45.1+ WT cells in the same thymus (open circles).

(F) Flow cytometric histograms of Ki67 staining in all

thymic NKT cells (top panel) or in the indicated NKT

cell subsets (bottom panel), showing concatenated data

for hpls/hpls and WT cells in the thymus from all four

mice shown in (E). (G) Percentage of NKT cells in the

indicated organs in mixed bone marrow chimeras ana-

lyzed separately for CD45.1+ +/+ and CD45.2+ hpls/hpls

cells. Statistics were calculated using the Mann–Whitney

U test. *p , 0.05, **p , 0.005.

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that the CD42CD8+ phenotype was indeed cell intrinsic (Fig. 2C)and also indicated that hpls/hpls NKT cells had a competitivedevelopmental advantage as can be seen by a higher ratio of thesecells as a total population (Fig. 2D). Analysis of the maturationalstages revealed that this bias was not detected at stage 1 of NKTdevelopment (CD442NK1.12) but was first apparent at stage 2(CD44+NK1.12) and maintained at stage 3 (CD44+NK1.1+) ofNKT cell development (Fig. 2D). This bias toward hpls/hplsNKT cells with maturation appeared to be at least partly due tohyperproliferation of stage 3 cells, as indicated by high-frequencystaining with Ki67 compared with the resting state of the corre-sponding WT cells in the same thymus (Fig. 2E). The highest levelof Ki67 staining was associated with the CD8+ NKT cell fraction(Fig. 2F). Thus, these data demonstrate that ZBTB7B plays anintrinsic role in the regulation of NKT cell development thatseems to be first manifest after positive selection in the thymus asthese cells begin to mature (stage 2). Analysis of NKT cells in theperipheral tissues of mixed bone marrow chimeras showed that thehpls/hpls NKT cells had a cell-intrinsic competitive advantage in allanalyzed organs including thymus, spleen, liver, and LN (Fig. 2G).Other cell surface markers including CD62-L and NK cell receptors

Ly6C, NKG2A/C/E, Ly49C/I, and NKG2D were also differentlyexpressed by the NKT cells in hpls/hpls mice (Fig. 3). The lowerlevels of NK1.1 observed on thymic hpls/hpls NKT cells was notobserved on hpls/hpls NKT cells from spleen or liver. Similarly,hpls/hpls NKT cells also had lower levels of Ly6C and the NKG2receptors in the thymus but higher levels in spleen and livercompared with WT NKT cells. Mutant NKT cells also tendedtoward higher expression of CD62-L, especially in spleen, pos-sibly explaining the increased percentage of NKT cells observedin LN of hpls/hpls mutant mice.

Zbtb7b mutant NKT cells preferentially develop into anIL-17–producing subset

A recent study (16) suggested that NKT cells from ZBTB7B-deficient mice were functionally impaired with much lower cy-tokine production compared with their WT counterparts, whichwas surprising given their otherwise mature phenotype. Ourfindings were consistent with this for the cytokines previouslytested (IFN-g, IL-4). Analysis of cytokine production by these cells,using intracellular cytokine staining following in vitro stimulation,revealed that very few mutant NKT cells produced IFN-g or TNF,

whereas a high proportion of NKT cells produced IL-17, whichwas the opposite of WT NKT cells (Fig. 4A). Given that hpls/hplsNKT cells clearly were functional and capable of cytokine pro-duction, we used cytometric bead array to test cytokine productionby these cells more comprehensively. Purified NKT cells werestimulated in three different ways: plate-bound CD3 and CD28,plate-bound CD1d loaded with a-GalCer, or spleen-derived den-dritic cells loaded with a-GalCer, and supernatants were harvestedafter 24 h. This revealed that cytokine production by hpls/hplsNKT cells, with the exception of IL-17, was drastically reduced,to the extent that many cytokines were near or below the detectionlimit (Fig. 4B). In some experiments, we also compared DN andCD8+ NKT cells from hpls/hpls mice and observed similar cyto-kine production regardless of the expression of CD8 (data notshown) suggesting that ZBTB7B acts at multiple levels on NKTcell development. This suggests that ZBTB7B is important inregulating the developmental balance of IL-17–producing NKTcells, which have recently been identified as a distinct subset ofNKT cells (known as NKT-17 cells) that make lower amounts ofother cytokines and have unique functions in vivo (4, 7).

IL-17–producing hpls/hpls NKT cells are RORgt+

The production of IL-17 is usually dependent on the transcriptionfactor RORgt, (28) and previous studies showed that IL-17–pro-ducing NKT cells express RORgt and the receptor for IL-23 (4, 7,29). To test whether hpls/hpls NKT cells overexpress either ofthese molecules, we performed real-time PCR for IL-23R andRORgt and found both to be increased in hpls/hpls NKT cells(Fig. 5A). The hpls/+ heterozygous mice showed a small in-crease in expression compared with the WT NKT cells. We alsotested for the transcription factors RUNX1 and RUNX3, whichhave been shown to play an important role in the regulation ofCD4 and CD8 coreceptor expression in conventional T cells (30),and the transcription factor T-bet, which is essential for progres-sion from the NK1.12 to the NK1.1+ stage of NKT development(31). No clear difference was observed for Runx1 and Runx3expression, but expression of T-bet was ∼3-fold reduced in thehpls/hpls mutant NKT cells. RT-PCR is unable to distinguishbetween an increased frequency of positive cells and increasedexpression per cell. To test for this directly and simultaneouslydetermine if the increased expression of IL-17 in Zbtb7b mutantNKT cells coincides with RORgt expression, NKT cells from

FIGURE 3. Altered expression of surface markers on NKT cells of Zbtb7bhpls/hpls mice. (A) Expression of the indicated surface markers and inhibitory and

activating NK receptors on NKT cells in the thymus, spleen, and liver was measured by flow cytometry. Histograms show concatenated samples from all mice

shown in (B). (B) Each dot represents an individual mouse; the bar graph shows the mean of all samples. T, S, and L denote thymus, spleen, and liver, respectively.

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+/+, hpls/+, and hpls/hpls mice were stimulated with PMA andionomycin and intracellular staining used to assess RORgt and IL-17 expression by flow cytometry. This revealed that ∼80% of themutant NKT cells in the thymus were positive for RORgt andabout two-thirds of those produced IL-17 (Fig. 5B–D). For spleen-derived NKT cells, the percentage of RORgt+ NKT cells was 20%in the hpls/hpls animals, and again, approximately two-thirds ofthese produced IL-17. In contrast, WT NKT cells were only 3–8%RORgt+, whereas heterozygous mice showed an intermediate phe-notype with around 20% of NKT cells in thymus expressing RORgt(Fig. 5C, 5D). Analysis of NKT cells in the thymus and liver ofmixed bone marrow chimeras showed that the increased expressionof RORgt and production of IL-17 in hpls/hpls NKT cells was cellintrinsic (Supplemental Fig. 3). Notably, the RORgt+ NKT cells hada lower expression of NK1.1 than RORgt2 NKT cells (Fig. 5B).Because this is similar to IL-17–producing NKT cells (NKT-17 cells)in WT mice (4, 7), this most likely does not reflect a specific effect ofthe mutant ZBTB7B protein on NK1.1 expression, but rather thepredominance of an NK1.1low IL-17–producing NKT subset witha specific phenotype. This interpretation is also supported by the

mutually exclusive expression of the transcription factors T-bet andRORgt as observed by dual labeling of NKT cells (Fig. 5E) and theexpression of IL-23R by hpls/hpls NKT cells (Fig. 5A), which isalso a marker of NKT-17 cells in WT mice (7, 29). To explore thispossibility further, we examined hpls/hpls NKT cells for CD103and CCR6 expression because high expression of these markers isassociated with NKT-17 cells in LN (32). Indeed, we found that theRORgt+ LN NKT subset from both WT and hpls/hpls mice wasCD103hi and CCR6hi, whereas the RORgt2 NKT subsets wereheterogeneous for these markers (Supplemental Fig. 2). This isconsistent with the previous study where IL-17 was only producedby a subset of CD103hi, CCR6hi, and RORgthi LN NKT cells (32).Taken together, the findings in this study strongly suggest that

NKT cell lineage decisions, and specifically the ratio of NKT-17cells to other NKT cells, is intrinsically regulated by the tran-scription factor ZBTB7B.

DiscussionAlthough several factors that control NKT cell numbers have beenidentified (33), there is little understanding of what controls the

FIGURE 4. Altered cytokine production by thy-

mic ZBTB7B mutant NKT cells. (A) Thymocytes

from ZBTB7B mutant and WT mice were activated

for 2.5 h with PMA and ionomycin and stained for

surface markers followed by intracellular staining

with Abs to IL-17, IFN-g, and TNF or with isotype

control Abs. All plots are gated on NKT cells

(TCRb+, a-GalCer–CD1d tetramer+). Gates for

cytokine-producing cells were set on isotype control

stains, and the numbers show the percentage of

NKT cells within these gates. Data for IL-17 are

from three different experiments on a pure C57BL/6

background. Data for IFN-g production are from

two independent experiments, with mice used in

experiment 1 from a pure C57BL/6 background,

whereas mice for experiment 2 were on a mixed

CBA 3 C57BL/6 background. (B) Sorted thymic

NKT cells were stimulated with 10 mg/ml anti-CD3

and anti-CD28 (first row), plate-bound CD1d and

a-GalCer (second row) or sorted splenic dendritic

cells and a-GalCer (third row). After 24 h, the su-

pernatant was assayed for the concentration of the

indicated cytokines, determined by a cytometric bead

array. Concentrations are in picograms/milliliter,

represented on a logarithmic scale. Values below 1

pg/ml were given a baseline value of 1. The results

are derived from four to 10 separate cultures col-

lected over two to three independent experiments.

Bars depict mean of all samples collected.

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development of distinct subsets of NKT cells. In this study, wedemonstrate that although ZBTB7B is not required for the de-velopment of normal numbers of NKT cells, it plays a critical rolein genetically predetermining their differentiation into differentsubsets defined by patterns of cell surface markers and cytokineproduction.It has been appreciated, almost since their discovery, that

NKT cells in mice and humans can be divided into subsets based onCD4 and CD8 expression. Furthermore, there are clear differences

in cytokine production by CD4+ and CD42 subsets of human NKTcells, where CD4+ cells produced both Th1 and Th2 type cyto-kines while CD42 NKT cells produced predominantly Th1 typecytokines (5, 6). Moreover, human CD8+ NKT cells may also befunctionally distinct from CD4+ and DN NKT cells (34). Thus, thecell surface phenotype of NKT cells appears to correlate withimportant functional diversity. While it is also clear that mouseNKT cells include diverse subsets defined by cell surface markersincluding CD4, there are some important distinctions with human

FIGURE 5. RORgt expression in Zbtb7b mutant NKT cells. (A) Expression of Zbtb7b, RORgt, IL23R, Runx1, Runx3, and Tbet mRNA in sorted thymic

NKT cells from Zbtb7b mutant, heterozygous, and WT mice was measured by RT-PCR. (B and C) Thymocytes and splenocytes from Zbtb7b mutant and

WT mice were activated for 3 h in the presence of GolgiStop (BD Pharmingen) with PMA and ionomycin and stained for surface markers followed by

intracellular staining with Abs recognizing IL-17 and the transcription factor RORgt. All plots are gated on NKT cells (TCRb+ or CD3+, PBS57–CD1d

tetramer+). (B) Representative FACS plots. (C) Percentages of RORgt+ NKT cells in thymus, spleen, liver, and LN. Data are representative of two

experiments (thymus, liver, and spleen) or one experiment (LN) with two to five mice (C57BL/6 background) per group. Data for heterozygous mice for

spleen and liver are from a single experiment. (D) Percentage of IL-17+ cells out of all RORgt+ NKT cells in the thymus. (E) Relative expression of RORgt+

and T-bet in individual NKT cells.

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NKT cells. There is no clear difference in cytokine productionbetween mature mouse CD4+ and CD42 NKT cells; both canmake Th1 and Th2 type cytokines (4). Also, mouse NKT cells donot normally include a population of CD8+ cells. However, mouseNKT cells can be subdivided into functionally distinct subsetsbased on the expression of NK1.1, and NK1.12 mouse NKT cellsproduce a distinct array of cytokines compared with NK1.1+

NKT cells, including higher IL-4 and lower IFN-g (4). Of particularinterest, a subset of CD42NK1.12 mouse NKT cells predominantlyproduce the proinflammatory cytokine IL-17 but not other cyto-kines (4, 7). This subset is thought to represent a distinct lineageof NKT cells, whose development depends on the transcriptionfactor RORgt (4, 7, 35). The production of IL-17, a proin-flammatory cytokine, imbues this subset of NKT cells withmarkedly different functional potential. IL-17–producing NKTcells have been associated with induction of airway neutrophilia(7), ozone-induced airway hyperreactivity (36), and collagen-induced arthritis (a model for rheumatoid arthritis) in mice (37).It is also likely, given the unique functions of IL-17 in autoim-munity and anti-microbial immunity (38), that IL-17–producingNKT cells will have different functions from non–IL-17–pro-ducing NKT cells in other disease settings.In contrast to normal WT mice, Zbtb7b mutant mice lack CD4+

NKT cells and instead harbor a population of CD8+ NKT cells(16, 17). Furthermore, antigenic stimulation of these cells revealeda major defect in IL-4 and IFN-g production, which led to thesuggestion that these cells were functionally hyporesponsive toTCR stimulation (16). However, IL-17 was not tested in that study,and our findings present a very different interpretation for the roleof ZBTB7B in NKT cell development, showing that althoughthese cells are deficient in IFN-g and IL-4 as published (16), theyare clearly capable of producing high levels of IL-17. Moreover,IL-17–producing Zbtb7b mutant NKT have lower levels of NK1.1,and they express high levels of RORgt and IL-23R, which alsoclosely aligns them with IL-17+ NKT cells in WT mice. Fur-thermore, both CD82 and CD8+ NKT cells in Zbtb7b mutant miceproduced IL-17, indicating that the CD8 phenotype is not directlyrelated to the altered cytokine profile and may reflect multipledevelopmental checkpoints that are controlled by ZBTB7B. Ourdata suggest that the NKT-17 lineage may be a default pathway forNKT cell development and that ZBTB7B is a key transcriptionfactor that drives the development of other phenotypically andfunctionally distinct NKT cell subsets. In support of this, T-bet,a transcription factor that is known to be critical for maturation ofIFN-g–producing NK1.1hi NKT cells (31), was present at muchreduced levels in Zbtb7b mutant NKT cells. At present, it is un-clear if ZBTB7B regulates the balance of development of theseNKT cell subsets by directly binding to the Rorc gene to influenceexpression of RORgt or if ZBTB7B somehow affects signalingthrough STAT3, another transcription factor required for the de-velopment of IL-17–producing T cells (39). Further studies in-cluding chromatin immunoprecipitation assays are required todifferentiate between these possibilities, but given the dramaticfunctional difference between NKT-17 cells and other NKT cells,identification of ZBTB7B as a major switch factor represents animportant step toward being able to control NKT cell function.The developmental and functional basis for CD4 and CD8

coreceptor expression by NKT cells has been a long-standingpuzzle in the field. Because NKT cell TCRs are CD1d re-stricted, there is no obvious role for CD4 or CD8 in binding toMHCclass II orMHC class I, respectively, nor is it clear how or why thesemolecules are modulated during NKT cell development. Our datausing “helpless” Zbtb7b mutant mice sheds new light on thisproblem, demonstrating that ZBTB7B is critical for development

and/or maintenance of CD4+ NKT cells, but it also inhibits theemergence of CD8+ NKT cells. This seems to be regulated atmultiple levels in a dose-dependent manner, because hpls/+ micehave diminished CD4+ NKT cells but do not have increased CD8+

NKT cells. The altered ratio of CD4+ and CD8+ NKT cells may beat least partly related to proliferative differences in the thymus,where mutant NKT cells that are DN and CD8+ proliferate ata higher rate than WT CD4+ or DN NKT cells, and our analysis ofmixed bone marrow chimeras shows that this effect is cell in-trinsic. However, proliferation is unlikely to explain fully thedifferences because CD8+ NKT cells are simply nonexistent in theperiphery of normal WT mice. Importantly, our study demon-strates that CD8 is gradually acquired by Zbtb7b mutantNKT cells, rather than by a failure to downregulate this surfacemarker after NKT cell selection from DP precursors in the thy-mus. This is further supported by the fact that many of these cellsare CD8a+CD8b2 in contrast to DP thymocytes that express bothCD8a and CD8b. An early study demonstrated that forced (trans-genic) expression of CD8 by all T cells resulted in depletion ofNKT cells (40), which suggested that mouse NKT cells that ex-press CD8 are deleted in the thymus, perhaps due to enhancedsignaling via CD8 resulting in negative selection. However, amore recent publication showed that thymocytes from homozy-gous CD8 transgenic mice have a shorter life span, which affectstheir ability to undergo distal TCR Va-Ja gene rearrangements(16). This makes secondary TCR rearrangements, required to in-corporate distal TCR-Ja genes such as Ja18 into the TCR-a-chain(41–44), less likely. This study also demonstrated that CD8 doesnot detectably bind to CD1d (16). Thus, the conclusion mostconsistent with our data are that the expression of ZBTB7B isnormally activated at a very early stage in NKT cell development,and in the absence of this transcription factor, CD8 expression isreacquired during NKT cell maturation. This is an intriguingconsideration in light of the existence of human CD8+ NKT cells.In the future, it will be important to determine whether human CD8+

NKT cells have lower amounts of ZBTB7B and express RORgt.The regulation of CD4 expression by conventional ab T cells

involves binding of RUNX1 and RUNX3 to the Cd4 silencer at thedifferent stages during their development to suppress the expres-sion of CD4 at the DN stage of thymic development or in CD8+

cytotoxic T cells (45). Furthermore, the expression of Zbtb7b inCD8+ cytotoxic T cells is silenced by Runx proteins (46), andRUNX1 has also been shown to enhance the expression of CD8 bycytotoxic T cells (41). But consistent with previous reports showingan essential role for RUNX1 in the development of NKT cells (41),we observed a comparable expression for both Runx1 and Runx3mRNA in Zbtb7b mutant and WT NKT cells.There are some similarities between our findings and those

previously reported in GATA-3–deficient mice (12), primarily alack of CD4+ NKT cells and reduced IFN-g production byNKT cells after TCR ligation. GATA-3 is known to bind theZbtb7b locus and is thought to promote Zbtb7b expression inNKT cells (17). However, GATA-3–deficient mice lack stage 2(CD44hiNK1.1low) NKT cells in the thymus (12) and exhibit adeficiency in peripheral NKT cells, which clearly distinguishesthis defect from that observed in Zbtb7b mutant NKT cells. Fur-thermore, NK1.1 was not lower in GATA-3–deficient NKT cells,they were able to produce IFN-g, and they do not express CD8(17). Thus, there appears to be at least two factors (GATA-3 andZBTB7B) that are important for controlling CD4 expression byNKT cells, and they work in a nonredundant manner.The precise mechanisms by which ZBTB7B regulates the de-

velopment of NKT cell subsets and how the Leu102Arg mutationaffects the function of the protein remain to be determined. It is

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currently unclear if the L102R mutation is a null allele or hasa dominant-negative effect, but as the number of RORgt+, IL-17–producing cells is intermediate in the heterozygous mice (Figs.4A, 5B), this appears to support the concept that the mutation ishaving a null effect. This also fits well with the fact that theLeu102Arg mutation is in the dimerization domain (SupplementalFig. 1) and very likely interferes with the dimerization ofZBTB7B required for transcriptional activation, and this likelyresults in a null allele in the homozygous state. Furthermore,based on our observations from hpls/+ and hpls/hpls mice, thedata suggest that full expression of this protein is required tomaintain normal numbers of CD4+ NKT cells because these arediminished (but not absent) in the heterozygous mice. In contrast,heterozygous mice showed no apparent defect in the percentage ornumber of conventional CD4+ T cells in the periphery. It was onlyin the homozygous mutant mice that we found an absence ofCD4+ NKT cells and an abundance of CD8+ NKT cells, sug-gesting that partial expression of ZBTB7B is still sufficient toprevent CD8+ NKT cells. Whether this simply represents a doseeffect acting at the same point in development or independenteffects at different stages in development is less clear. However,the fact that CD8+ NKT cells were far more abundant in spleenand liver compared with thymus supports the second scenario,where an absence of functional ZBTB7B allows a delayed in-crease in CD8+ NKT cells, which does not occur in the presenceof suboptimal ZBTB7B expression. Given the large variability inthe population size of preexisting NKT cells especially in humans,variability in this process may contribute to individual variabilityin the types of immune responses made to common Ags. Futurefunctional studies comparing WT and hpls/hpls NKT cells, usingadoptive transfer into NKT cell–deficient mice (containing normalnumbers of conventional CD4 T cells), are required to study theinfluence of the hpls mutation on NKT cells in disease modelssuch as infection, cancer, and autoimmunity.In summary, we have determined that ZBTB7B is a critical factor

controlling the development of NKT cell subsets defined by cellsurface CD4 and CD8 expression, where CD4 expression is tightlyregulated by ZBTB7B such that even partial reduction of thisfactor diminishes CD4+ NKT cells, while the emergence of CD8+

NKT cells only occurs in the absence of ZBTB7B. ZBTB7B alsodetermines the functional potential of NKT cells, such that inits absence, an RORgt+ IL-23R+ IL-17+ (NKT-17) phenotypepredominates. This study demonstrates that the ZBTB7B tran-scription factor has a profound impact on the development andfunctional potential of NKT cells. Further studies into the ZBTB7B-mediated molecular pathways that culminate in the altered pheno-types are required.

AcknowledgmentsWe thank Owen Siggs and Lina Tze for suggesting Zbtb7b as a candidate

gene and Belinda Whittle and the genotyping team of the Australian Phe-

nomics Facility for genotyping, Ken Field (University of Melbourne) and

Natalie Saunders (St. Vincent’s Institute) for assistance with flow cytomet-

ric cell sorting, and we acknowledge the support of the National Institutes

of Health Tetramer Facility for providing the PBS57-loaded CD1d tetra-

mer for some of the experiments.

DisclosuresThe authors have no financial conflicts of interest.

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