ZBTB7B (Th-POK) Regulates the Development of IL-17−Producing CD1d-Restricted Mouse NKT Cells

This information is current as Anselm Enders, Sanda Stankovic, Charis Teh, Adam P. of September 29, 2021. Uldrich, Mehmet Yabas, Torsten Juelich, John A. Altin, Sandra Frankenreiter, Hannes Bergmann, Carla M. Roots, Konstantinos Kyparissoudis, Chris C. Goodnow and Dale I. Godfrey

J Immunol 2012; 189:5240-5249; Prepublished online 26 Downloaded from October 2012; doi: 10.4049/jimmunol.1201486 http://www.jimmunol.org/content/189/11/5240 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/10/26/jimmunol.120148 Material 6.DC1 References This article cites 46 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/189/11/5240.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ZBTB7B (Th-POK) Regulates the Development of IL-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 and Dale 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 Downloaded from 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 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- http://www.jimmunol.org/ ing the balance of effector subsets within the NKT cell population. The Journal of Immunology, 2012, 189: 5240–5249.

he factors that regulate formation of distinct subsets factors is important to explain individual variability in physio- of effector T cells are not well understood. While these logical or pathological immune reactions to common Ags. T responses are clearly influenced by the nature and route of NKT cells are CD1d-restricted, glycolipid Ag-reactive T cells exposure of an encountered Ag, genetic wiring also influences the that represent a unique population of effector T cells in mice kinds of effector T cell responses. Understanding these genetic and humans. These cells express a heavily biased TCR repertoire, composed of an invariant TCR a-chain (Va14Ja18 in mice, by guest on September 29, 2021 *Ramaciotti Immunization Genomics Laboratory, Department of Immunology, John Va24Ja18 in humans) paired with a limited array of TCR Curtin School of Medical Research, Australian National University, Canberra, Aus- b-chains (Vb8.2, Vb7, or Vb2 in mice, Vb11 in humans) (1, 2). † tralian Capital Territory 0200, Australia; Department of Microbiology and Immu- NKT cells can influence a broad spectrum of diseases, ranging nology, University of Melbourne, Parkville, Victoria 3010, Australia; ‡Department of Immunology, John Curtin School of Medical Research, Australian National Univer- from suppression of autoimmune diseases like type 1 diabetes, to sity, Canberra, Australian Capital Territory 0200, Australia; and xAustralian Phenom- promotion of immunity, to cancer and infection (3). This paradox- ics Facility, Australian National University, Canberra, Australian Capital Territory ical ability to promote or suppress immune responses is associated 0200, Australia with the profound ability of NKT cells to produce a spectrum of 1D.I.G. and C.C.G. contributed equally to this work. cytokines within hours of stimulation. At the population level, Received for publication May 29, 2012. Accepted for publication September 26, 2012. NKT cells produce seemingly antagonistic cytokines including IFN- g, IL-4, IL-10, IL-13, and IL-17, although NKT cells can be divided This work was supported by research grants from the Wellcome Trust, the National Institutes of Health (AI054523), the National Health and Medical Research Council into functionally distinct subsets that are capable of preferentially of Australia (NHMRC), the CASS Foundation, and the Ramaciotti Foundations. A.E. producing only some of these cytokines (4–7), which may partly was supported by a Deutsche Forschungsgemeinschaft Research Fellowship (EN 790/1-1) and an NHMRC Project Grant (APP1009190) and Career Development explain the diverse functional outcomes associated with these cells. Fellowship (APP1035858). D.I.G. was supported by an NHMRC Senior Principal Human NKT cells vary widely in frequency between individuals, Research Fellowship (1020770). C.C.G. was supported by an NHMRC Australia yet are stable within individuals (8, 9). Human NKT cells include Fellowship (585490) and by an Australian Research Council Federation Fellowship. CD4+,CD42CD82 (double negative [DN]), and CD8+ subsets, and 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 the each of these exhibit distinct cytokine profiles, which again suggests experiments. that they have distinct functions in vivo. The ratio of CD4/CD8 de- The funders of the study had no role in study design, data collection and analysis, fined subsets of NKT cells also varies widely between individuals decision to publish, or preparation of the manuscript. (10). Given this variability, combined with their powerful immuno- Address correspondence and reprint requests to Prof. Dale I. Godfrey or Prof. Chris regulatory potential, it is very important to decipher the factors that C. Goodnow, Department of Microbiology and Immunology, University of Mel- regulate NKT cell development and homeostasis, including factors bourne, Parkville, VIC 3010, Australia (D.I.G.) or Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT that determine the balance of functionally distinct NKT cell subsets. 0200, Australia (C.C.G.). E-mail addresses: [email protected] (D.I.G.) and Many molecules, including cell surface receptors, signal trans- [email protected] (C.C.G.) duction and transcription factors, have been identified that selec- The online version of this article contains supplemental material. tively regulate NKT cell numbers independently from conventional Abbreviations used in this article: DN, double negative; LN, lymph node; RORgt, T cells (11). For example, the SLAM/SAP/fyn signaling pathway retinoic acid–related orphan receptor gt; WT, wild-type. is selectively important for NKT cell development while dispens- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 able for T cell development in the thymus (11). However, little is www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201486 The Journal of Immunology 5241 known about what regulates the differentiation of NKT cell sub- MEM nonessential amino acids (Life Technologies), and 5.5 mM2-mer- sets. One study, an investigation of NKT cell development in captoethanol. Stimulated cells were then washed, stained for surface GATA-3 knockout mice, provided data showing that CD4+ markers, and stained intracellularly using FITC-conjugated anti-mouse IL-17A (BioLegend) or isotype-matched control Abs (BD Pharmingen), NKT cells were preferentially inhibited in the absence of this RORgt–PE, or allophycocyanin (eBioscience) using the eBioscience factor (12). What factors regulate the appropriate expression of the fixation/permeabilization kit. CD4 or CD8 coreceptors in MHC class II– or MHC class I–re- For cytometric bead array, thymocytes were pooled from several mice per stricted thymocytes has itself been a long-standing issue. In 2005, group and enriched by either staining with anti-CD24 (J11D) followed by depletion using rabbit complement (C-SIX Diagnostics) in the presence two studies showed that the ZBTB7B (previ- of DNase (Roche Diagnostics) or by staining cells with PE-conjugated ously called Th-POK and cKrox) plays a key role in maintaining CD1d–a-GalCer tetramer and subsequent incubation with anti-PE CD4 expression in MHC class II–restricted thymocytes (13, 14). microbeads (Miltenyi Biotech). Labeled cells were then enriched by ZBTB7B promotes CD4 expression indirectly by preventing passing them through a magnetic column and were further stained for flow Runx1- and Runx3-mediated downregulation of CD4 in conven- cytometric purification. This second method was also used to enrich splenic dendritic cells (based on CD11c expression). Enriched cells were tional TCRab T cells (15). It has been shown subsequently that sorted using a FACSAria (BD Biosciences) in the Department of Micro- CD4 expression by NKT cells is also dependent on ZBTB7B (16, biology and Immunology Flow Cytometry Facility (University of Mel- 17). Furthermore, in the absence of ZBTB7B, NKT cell cytokine bourne) to obtain highly purified populations. Sorted NKT cells were 3 4 production was impaired, which led to the suggestion that ZBTB7B stimulated by placing them in 96-well plates (2 10 cells/well), coated with anti-CD3 and anti-CD28, or with soluble CD1d loaded with is required for full NKT cell maturation and activation (16). a-GalCer, or by coculture with sorted splenic dendritic cells (1 3 104 In this study, we describe a novel mouse strain, termed “help- dendritic cells/well) loaded with a-GalCer. After 24 h, the supernatant was less,” carrying a point mutation in Zbtb7b creating a single amino collected and the concentration of cytokines secreted into the medium Downloaded from acid substitution in the BTB-POZ domain. Using this mouse determined by cytometric bead array (BD Pharmingen). model, we show that ZBTB7B plays an essential, cell intrinsic, Cell sorting, RNA extraction, cDNA synthesis, and quantitative and dose-dependent role in establishing the balance of different PCR NKT cell subsets, including maintenance of CD4, inhibition of CD8 expression, and development of the retinoic acid–related Single-cell suspensions from thymocytes were prepared and stained as for + flow cytometric analysis. Samples were sorted on a FACSAria sorter (BD orphan receptor gt(RORgt)-positive IL-17 population of NKT cells. Biosciences) at the Flow Cytometry Facility of the John Curtin School http://www.jimmunol.org/ ZBTB7B thereby establishes a genetically predetermined profile of of Medical Research (Australian National University). Total RNA was CD1d-restricted NKT cells. extracted using RNA TRIzol reagent (Molecular Research Centre) and reverse transcribed using random oligonucleotide primers and 50 U Superscript II reverse transcriptase (Invitrogen) as detailed in the Materials and Methods manufacturer’s guidelines. SYBR Green real-time PCR reactions were Mice performed in 96-well plates (PerkinElmer) with an ABI PRISM 7900 Real-

hpls/hpls Time System (PerkinElmer/PE Biosystems) at the Biomolecular Resource The Zbtb7b strain derived from a C57BL/6 male treated three times Facility (John Curtin School of Medical Research, Australian National i.p. with 100 mg/kg N-ethyl-N-nitrosourea at weekly intervals. Mice were University). To correlate the threshold (Ct) values from the cDNA am- 3 maintained on a pure C57BL/6 background or on a mixed CBA C57BL/6 plification plots to fold differences between samples, the DDCt method was by guest on September 29, 2021 background. All mice were housed in specific pathogen–free conditions at applied using the housekeeping GAPDH. the Australian Phenomics Facility. All experimental procedures were ap- proved by the Australian National University Animal Ethics and Experi- Generation of bone marrow chimeras mentation Committee. B6.SJL CD45.1 mice were irradiated with 10 Gy and injected with 2 3 106 Sequencing and genotyping bone marrow cells consisting of a 50:50 mix of wild-type (WT) B6.SJL CD45.1+ and either WT or mutant C57BL/6 (CD45.2+) cells. They were All exons and splice sites of Zbtb7b were amplified, and primers for se- allowed to reconstitute for 8 wk before analysis. quencing were designed using Australian Phenomics Facility software to amplify all exons and splice sites for Zbtb7b. The amplification and dual sequence run were performed at the Brisbane node of the Australian Genome Results Research Facility. Sequence analysis was conducted at the Australian Phe- Identification of the Zbtb7b mutant strain helpless (hpls) nomics Facility using Lasergene software (DNAStar). A T to G substitu- tion of bp 480 was identified in exon 2 (ensmuse000001765423) of Zbtb7b, In a genome-wide screen for N-ethyl-N-nitrosurea–induced point resulting in a CTG (Leu) to a CGG (Arg) amino acid change. Mice were mutations affecting the development of the immune system (19), genotyped using an Amplifluor assay (Chemicon). All primer sequences we identified a strain that was deficient in CD4+ T cells in pe- are available on request. ripheral blood and spleen. Further analysis revealed a block in + dim Flow cytometry CD4 development at the CD4 CD8 stage in the thymus (Fig. 1A). This phenotype is identical to the phenotype described for the Cell suspensions from thymus and spleen were prepared by passing the cells HelperDeficient (HD) strain caused by an amino acid substitution through a cell strainer (BD Biosciences) or stainless steel sieve, followed by lysis of RBCs for spleen and liver samples. Liver lymphocytes were in the DNA-binding zinc finger domain of the transcription factor isolated by centrifugation over a Percoll gradient. Cell suspensions were ZBTB7B, previously called Th-POK or cKrox (13, 20) or labeled with fluorochrome-coupled Abs according to standard protocols knockout mice with a Zbtb7b null allele (21). Because of these and run on an LSR II or Canto flow cytometer (BD Biosciences) followed similarities, we sequenced Zbtb7b and identified a mutation by analysis with FlowJo (Tree Star). a-GalCer–loaded CD1d tetramers were produced in house, using a mouse CD1d baculovirus construct originally changing a conserved leucine to arginine in the BTB-POZ domain provided by Prof. Mitchell Kronenberg, as previously described (18). For (Fig. 1B). BTB-POZ domains mediate homodimerization and some experiments, a-GalCer (PBS57)-loaded CD1d tetramers provided by heterodimerization, association with nuclear corepressors, and the National Institutes of Health Tetramer Facility were used. ubiquitination (22). Deletion of the BTB-POZ domain from a In vitro stimulation and cytokine analysis Zbtb7b transgene inactivated its ability to deviate MHC class I– restricted T cells into the CD4 lineage (14). The leucine that is For the intracellular cytokine staining assay, cells were stimulated with 50 mutated in helpless mice is buried within the homologous PZLF ng/ml phorbol ester and 500 ng/ml ionomycin for 2.5–3.5 h at 37˚C in the BTB-POZ domain (Supplemental Fig. 1 and Ref. 23). The presence of monensin in RPMI 1640 culture media supplemented with L102R 10% heat-inactivated FCS, glutamine (Life Technologies), 10 mM sodium ZBTB7B disrupts CD4 cell differentiation as completely as pyruvate (Life Technologies), 10 mM HEPES (Life Technologies), 10 mM the null mutation, but whether this reflects misfolding of the BTB 5242 DEVELOPMENT OF IL-17 PRODUCING NKT-CELLS DEPENDS ON ZBTB7B Downloaded from http://www.jimmunol.org/

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 by guest on September 29, 2021 domains from the indicated , 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)ofZbtb7bhpls/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)ofZbtb7bhpls/hpls, Zbtb7bhpls/+, and Zbtb7b+/+ mice. Each data point represents a different mouse, and the bars represent the mean. (F)PercentageofCD4+,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. domain or destabilization of the ZBTB7B as a whole or NKT cells express CD4, and none are CD8+. In the helpless strain, loss of particular protein–protein interactions is unclear. Geno- this was completely reversed with ∼70–80% of NKT cells in the typing of affected and unaffected progeny from numerous helpless spleen expressing CD8, and none were CD4+ (Fig. 1C). carriers confirmed that the failure of CD4 cell differentiation was Examination of NKT cells in different tissues revealed that the inherited in complete concordance with the Zbtb7b mutation in a percentage in thymus, spleen, and liver was comparable between recessive fashion. As noted previously for the HD strain (24), ho- WT and hpls/hpls mice, whereas the percentages and absolute cell mozygous affected mice on the parental C57BL/6 background were numbers of these cells in blood, bone marrow, and lymph nodes born at around half the expected frequency, and affected mice (LNs) were clearly higher (Fig. 1D, 1E). Notably, Zbtb7b het- showed poor breeding efficiency. This was rescued by keeping the erozygous mice also showed an increased percentage of NKT cells mice on a mixed C57BL/6 3 CBA background, where homozygotes in thymus, LN, and bone marrow, but not in spleen and liver (Fig. were obtained at Mendelian ratios. The embryonic lethality on 1D). The CD8+ NKT cell phenotype was detectable in thymus of the C57BL/6 background may indicate essential functions for hpls/hpls mice (∼20% CD8+) but far more pronounced in the ZBTB7B in other processes such as collagen gene regulation (25). spleen and liver where ∼80% were CD8+ (Fig. 1F). Further analysis It was previously reported that CD4 expression by NKT cells is of the CD8+ NKT cells in thymus, spleen, and liver showed that disrupted in Th-POK–deficient mice (16, 17), so we first investi- they included some cells that were CD8a+b2 and some that gated whether the Zbtb7b point mutation in hpls/hpls mice had were CD8a+b+ (Fig.1G).Furthermore,heterozygoushpls/+ a similar impact on these cells. Normally, around 70% of spleen mice exhibited an intermediate phenotype, where CD4+ NKT The Journal of Immunology 5243 cells were reduced, yet there was no sign of CD8 upregulation, in those in WT mice, this suggested there was no major problem with both thymus and periphery, thus hpls/+ mice were highly enriched the selection and expansion of these cells as a total population. for CD42CD82 NKT cells (Fig. 1F). This contrasts with the de- NKT cell development can be divided into three developmentally velopment of conventional CD4+ T cells that is seemingly unaf- distinct stages: stage 1 (CD44loNK1.12); stage 2 (CD44+NK1.12); fected in heterozygous mice (Fig. 1A) strongly suggesting that the stage 3 (CD44+NK1.1+) (26, 27); and these stages can be further effect on NKT surface marker expression is not due to a dominant- divided into CD4+ and CD42, a split that occurs at approximately negative effect of the hpls mutation. stage 2 (4). Although hpls/hpls NKT cells were mostly mature, as defined by their NK1.1+CD44hi phenotype, they expressed slightly NKT cell development is abnormal in Zbtb7b mutant mice lower levels of NK1.1 (Fig. 2A). Although CD8+ NKT cells were The developmental events giving rise to the unusual CD8+ detected in the hpls/hpls thymus, the high-intensity CD8 expres- NKT cell phenotype in Zbtb7b mutant mice have not been de- sion observed with hpls/hpls peripheral NKT cells (Fig. 1C) was termined; however, the accumulation of these cells in peripheral not reflected in the thymus where they were mostly CD8lo. This tissues at much higher levels than in thymus suggested this occurs suggested that the emergence of CD8+ NKT cells begins in the as a late event in NKT cell development. Therefore, we more thymus but is not fully manifested until these cells are in the carefully examined NKT cells in the thymus to determine the periphery. Examination of increasingly mature NKT cell subsets origins of this defect. Analysis of Zbtb7b mRNA expression by revealed that low CD8 expression was detectable from the earliest real-time PCR revealed that both immature NK1.12 and mature CD44loNK1.12 stage, but the percentage of CD8+ cells increased NK1.1+ NKT cells expressed Zbtb7b (data not shown). Zbtb7b as the NKT cells matured through CD44+NK1.12 and CD44+ expression levels were similar to CD4+CD8dim thymocytes, lower NK1.1+ stages (Fig. 2A, 2B). Downloaded from than total CD4+ single-positive thymocytes, but clearly above To determine whether the defects in NKT cell development in double-positive and CD8 single-positive thymocytes that have hpls/hpls mice were cell intrinsic, we performed mixed bone mar- expression at or just above background (13). Because NKT cell row chimera experiments to compare WT and hpls/hpls NKT cells numbers in the thymus of Zbtb7b mutant mice were comparable to developing in the same environment. These results demonstrated http://www.jimmunol.org/ 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- resentsanindividualmouse;dataarefromatleastfour by guest on September 29, 2021 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 marroworigininthesameanimal.(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)orintheindicatedNKT 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. 5244 DEVELOPMENT OF IL-17 PRODUCING NKT-CELLS DEPENDS ON ZBTB7B that the CD42CD8+ phenotype was indeed cell intrinsic (Fig. 2C) whereas a high proportion of NKT cells produced IL-17, which and also indicated that hpls/hpls NKT cells had a competitive was the opposite of WT NKT cells (Fig. 4A). Given that hpls/hpls developmental advantage as can be seen by a higher ratio of these NKT cells clearly were functional and capable of cytokine pro- cells as a total population (Fig. 2D). Analysis of the maturational duction, we used cytometric bead array to test cytokine production stages revealed that this bias was not detected at stage 1 of NKT by these cells more comprehensively. Purified NKT cells were development (CD442NK1.12) but was first apparent at stage 2 stimulated in three different ways: plate-bound CD3 and CD28, (CD44+NK1.12) and maintained at stage 3 (CD44+NK1.1+)of plate-bound CD1d loaded with a-GalCer, or spleen-derived den- NKT cell development (Fig. 2D). This bias toward hpls/hpls dritic cells loaded with a-GalCer, and supernatants were harvested NKT cells with maturation appeared to be at least partly due to after 24 h. This revealed that cytokine production by hpls/hpls hyperproliferation of stage 3 cells, as indicated by high-frequency NKT cells, with the exception of IL-17, was drastically reduced, staining with Ki67 compared with the resting state of the corre- to the extent that many cytokines were near or below the detection sponding WT cells in the same thymus (Fig. 2E). The highest level limit (Fig. 4B). In some experiments, we also compared DN and of Ki67 staining was associated with the CD8+ NKT cell fraction CD8+ NKT cells from hpls/hpls mice and observed similar cyto- (Fig. 2F). Thus, these data demonstrate that ZBTB7B plays an kine production regardless of the expression of CD8 (data not intrinsic role in the regulation of NKT cell development that shown) suggesting that ZBTB7B acts at multiple levels on NKT seems to be first manifest after positive selection in the thymus as cell development. This suggests that ZBTB7B is important in these cells begin to mature (stage 2). Analysis of NKT cells in the regulating the developmental balance of IL-17–producing NKT peripheral tissues of mixed bone marrow chimeras showed that the cells, which have recently been identified as a distinct subset of hpls/hpls NKT cells had a cell-intrinsic competitive advantage in all NKT cells (known as NKT-17 cells) that make lower amounts of Downloaded from analyzed organs including thymus, spleen, liver, and LN (Fig. 2G). other cytokines and have unique functions in vivo (4, 7). Other cell surface markers including CD62-L and NK cell receptors IL-17–producing hpls/hpls NKT cells are RORgt+ Ly6C, NKG2A/C/E, Ly49C/I, and NKG2D were also differently expressedbytheNKTcellsinhpls/hpls mice (Fig. 3). The lower The production of IL-17 is usually dependent on the transcription levels of NK1.1 observed on thymic hpls/hpls NKT cells was not factor RORgt, (28) and previous studies showed that IL-17–pro-

observed on hpls/hpls NKT cells from spleen or liver. Similarly, ducing NKT cells express RORgt and the receptor for IL-23 (4, 7, http://www.jimmunol.org/ hpls/hpls NKT cells also had lower levels of Ly6C and the NKG2 29). To test whether hpls/hpls NKT cells overexpress either of receptors in the thymus but higher levels in spleen and liver these molecules, we performed real-time PCR for IL-23R and compared with WT NKT cells. Mutant NKT cells also tended RORgt and found both to be increased in hpls/hpls NKT cells toward higher expression of CD62-L, especially in spleen, pos- (Fig. 5A). The hpls/+ heterozygous mice showed a small in- sibly explaining the increased percentage of NKT cells observed crease in expression compared with the WT NKT cells. We also in LN of hpls/hpls mutant mice. tested for the transcription factors RUNX1 and RUNX3, which have been shown to play an important role in the regulation of Zbtb7b mutant NKT cells preferentially develop into an CD4 and CD8 coreceptor expression in conventional T cells (30),

IL-17–producing subset and the transcription factor T-bet, which is essential for progres- by guest on September 29, 2021 A recent study (16) suggested that NKT cells from ZBTB7B- sion from the NK1.12 to the NK1.1+ stage of NKT development deficient mice were functionally impaired with much lower cy- (31). No clear difference was observed for Runx1 and Runx3 tokine production compared with their WT counterparts, which expression, but expression of T-bet was ∼3-fold reduced in the was surprising given their otherwise mature phenotype. Our hpls/hpls mutant NKT cells. RT-PCR is unable to distinguish findings were consistent with this for the cytokines previously between an increased frequency of positive cells and increased tested (IFN-g, IL-4). Analysis of cytokine production by these cells, expression per cell. To test for this directly and simultaneously using intracellular cytokine staining following in vitro stimulation, determine if the increased expression of IL-17 in Zbtb7b mutant revealed that very few mutant NKT cells produced IFN-g or TNF, NKT 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. The Journal of Immunology 5245

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 Downloaded from 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 http://www.jimmunol.org/ 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. by guest on September 29, 2021

+/+, hpls/+, and hpls/hpls mice were stimulated with PMA and mutually exclusive expression of the transcription factors T-bet and ionomycin and intracellular staining used to assess RORgt and IL- RORgt as observed by dual labeling of NKT cells (Fig. 5E) and the 17 expression by flow cytometry. This revealed that ∼80% of the expression of IL-23R by hpls/hpls NKT cells (Fig. 5A), which is mutant NKT cells in the thymus were positive for RORgt and also a marker of NKT-17 cells in WT mice (7, 29). To explore this about two-thirds of those produced IL-17 (Fig. 5B–D). For spleen- possibility further, we examined hpls/hpls NKT cells for CD103 derived NKT cells, the percentage of RORgt+ NKT cells was 20% and CCR6 expression because high expression of these markers is in the hpls/hpls animals, and again, approximately two-thirds of associated with NKT-17 cells in LN (32). Indeed, we found that the these produced IL-17. In contrast, WT NKT cells were only 3–8% RORgt+ LN NKT subset from both WT and hpls/hpls mice was RORgt+, whereas heterozygous mice showed an intermediate phe- CD103hi and CCR6hi, whereas the RORgt2 NKT subsets were notype with around 20% of NKT cells in thymus expressing RORgt heterogeneous for these markers (Supplemental Fig. 2). This is (Fig. 5C, 5D). Analysis of NKT cells in the thymus and liver of consistent with the previous study where IL-17 was only produced mixed bone marrow chimeras showed that the increased expression by a subset of CD103hi, CCR6hi, and RORgthi LN NKT cells (32). of RORgt and production of IL-17 in hpls/hpls NKT cells was cell Taken together, the findings in this study strongly suggest that intrinsic (Supplemental Fig. 3). Notably, the RORgt+ NKT cells had NKT cell lineage decisions, and specifically the ratio of NKT-17 a lower expression of NK1.1 than RORgt2 NKT cells (Fig. 5B). cells to other NKT cells, is intrinsically regulated by the tran- Because this is similar to IL-17–producing NKT cells (NKT-17 cells) scription factor ZBTB7B. in WT mice (4, 7), this most likely does not reflect a specific effect of the mutant ZBTB7B protein on NK1.1 expression, but rather the Discussion predominance of an NK1.1low IL-17–producing NKT subset with Although several factors that control NKT cell numbers have been a specific phenotype. This interpretation is also supported by the identified (33), there is little understanding of what controls the 5246 DEVELOPMENT OF IL-17 PRODUCING NKT-CELLS DEPENDS ON ZBTB7B Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

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. development of distinct subsets of NKT cells. In this study, we in cytokine production by CD4+ and CD42 subsets of human NKT demonstrate that although ZBTB7B is not required for the de- cells, where CD4+ cells produced both Th1 and Th2 type cyto- velopment of normal numbers of NKT cells, it plays a critical role kines while CD42 NKT cells produced predominantly Th1 type in genetically predetermining their differentiation into different cytokines (5, 6). Moreover, human CD8+ NKT cells may also be subsets defined by patterns of cell surface markers and cytokine functionally distinct from CD4+ and DN NKT cells (34). Thus, the production. cell surface phenotype of NKT cells appears to correlate with It has been appreciated, almost since their discovery, that important functional diversity. While it is also clear that mouse NKT cells in mice and humans can be divided into subsets based on NKT cells include diverse subsets defined by cell surface markers CD4 and CD8 expression. Furthermore, there are clear differences including CD4, there are some important distinctions with human The Journal of Immunology 5247

NKT cells. There is no clear difference in cytokine production and/or maintenance of CD4+ NKT cells, but it also inhibits the between mature mouse CD4+ and CD42 NKT cells; both can emergence of CD8+ NKT cells. This seems to be regulated at make Th1 and Th2 type cytokines (4). Also, mouse NKT cells do multiple levels in a dose-dependent manner, because hpls/+ mice not normally include a population of CD8+ cells. However, mouse have diminished CD4+ NKT cells but do not have increased CD8+ NKT cells can be subdivided into functionally distinct subsets NKT cells. The altered ratio of CD4+ and CD8+ NKT cells may be based on the expression of NK1.1, and NK1.12 mouse NKT cells at least partly related to proliferative differences in the thymus, produce a distinct array of cytokines compared with NK1.1+ where mutant NKT cells that are DN and CD8+ proliferate at NKT cells, including higher IL-4 and lower IFN-g (4). Of particular a higher rate than WT CD4+ or DN NKT cells, and our analysis of interest, a subset of CD42NK1.12 mouse NKT cells predominantly mixed bone marrow chimeras shows that this effect is cell in- produce the proinflammatory cytokine IL-17 but not other cyto- trinsic. However, proliferation is unlikely to explain fully the kines (4, 7). This subset is thought to represent a distinct lineage differences because CD8+ NKT cells are simply nonexistent in the of NKT cells, whose development depends on the transcription periphery of normal WT mice. Importantly, our study demon- factor RORgt (4, 7, 35). The production of IL-17, a proin- strates that CD8 is gradually acquired by Zbtb7b mutant flammatory cytokine, imbues this subset of NKT cells with NKT cells, rather than by a failure to downregulate this surface markedly different functional potential. IL-17–producing NKT marker after NKT cell selection from DP precursors in the thy- cells have been associated with induction of airway neutrophilia mus. This is further supported by the fact that many of these cells (7), ozone-induced airway hyperreactivity (36), and collagen- are CD8a+CD8b2 in contrast to DP thymocytes that express both induced arthritis (a model for rheumatoid arthritis) in mice (37). CD8a and CD8b. An early study demonstrated that forced (trans-

It is also likely, given the unique functions of IL-17 in autoim- genic) expression of CD8 by all T cells resulted in depletion of Downloaded from munity and anti-microbial immunity (38), that IL-17–producing NKT cells (40), which suggested that mouse NKT cells that ex- NKT cells will have different functions from non–IL-17–pro- press CD8 are deleted in the thymus, perhaps due to enhanced ducing NKT cells in other disease settings. signaling via CD8 resulting in negative selection. However, a In contrast to normal WT mice, Zbtb7b mutant mice lack CD4+ more recent publication showed that thymocytes from homozy- NKT cells and instead harbor a population of CD8+ NKT cells gous CD8 transgenic mice have a shorter life span, which affects

(16, 17). Furthermore, antigenic stimulation of these cells revealed their ability to undergo distal TCR Va-Ja gene rearrangements http://www.jimmunol.org/ a major defect in IL-4 and IFN-g production, which led to the (16). This makes secondary TCR rearrangements, required to in- suggestion that these cells were functionally hyporesponsive to corporate distal TCR-Ja such as Ja18 into the TCR-a-chain TCR stimulation (16). However, IL-17 was not tested in that study, (41–44), less likely. This study also demonstrated that CD8 does and our findings present a very different interpretation for the role not detectably bind to CD1d (16). Thus, the conclusion most of ZBTB7B in NKT cell development, showing that although consistent with our data are that the expression of ZBTB7B is these cells are deficient in IFN-g and IL-4 as published (16), they normally activated at a very early stage in NKT cell development, are clearly capable of producing high levels of IL-17. Moreover, and in the absence of this transcription factor, CD8 expression is IL-17–producing Zbtb7b mutant NKT have lower levels of NK1.1, reacquired during NKT cell maturation. This is an intriguing and they express high levels of RORgt and IL-23R, which also consideration in light of the existence of human CD8+ NKT cells. by guest on September 29, 2021 closely aligns them with IL-17+ NKT cells in WT mice. Fur- In the future, it will be important to determine whether human CD8+ thermore, both CD82 and CD8+ NKT cells in Zbtb7b mutant mice NKT cells have lower amounts of ZBTB7B and express RORgt. produced IL-17, indicating that the CD8 phenotype is not directly The regulation of CD4 expression by conventional ab T cells related to the altered cytokine profile and may reflect multiple involves binding of RUNX1 and RUNX3 to the Cd4 silencer at the developmental checkpoints that are controlled by ZBTB7B. Our different stages during their development to suppress the expres- data suggest that the NKT-17 lineage may be a default pathway for sion of CD4 at the DN stage of thymic development or in CD8+ NKT cell development and that ZBTB7B is a key transcription cytotoxic T cells (45). Furthermore, the expression of Zbtb7b in factor that drives the development of other phenotypically and CD8+ cytotoxic T cells is silenced by Runx proteins (46), and functionally distinct NKT cell subsets. In support of this, T-bet, RUNX1 has also been shown to enhance the expression of CD8 by a transcription factor that is known to be critical for maturation of cytotoxic T cells (41). But consistent with previous reports showing IFN-g–producing NK1.1hi NKT cells (31), was present at much an essential role for RUNX1 in the development of NKT cells (41), reduced levels in Zbtb7b mutant NKT cells. At present, it is un- we observed a comparable expression for both Runx1 and Runx3 clear if ZBTB7B regulates the balance of development of these mRNA in Zbtb7b mutant and WT NKT cells. NKT cell subsets by directly binding to the Rorc gene to influence There are some similarities between our findings and those expression of RORgt or if ZBTB7B somehow affects signaling previously reported in GATA-3–deficient mice (12), primarily a through STAT3, another transcription factor required for the de- lack of CD4+ NKT cells and reduced IFN-g production by velopment of IL-17–producing T cells (39). Further studies in- NKT cells after TCR ligation. GATA-3 is known to bind the cluding chromatin immunoprecipitation assays are required to Zbtb7b locus and is thought to promote Zbtb7b expression in differentiate between these possibilities, but given the dramatic NKT cells (17). However, GATA-3–deficient mice lack stage 2 functional difference between NKT-17 cells and other NKT cells, (CD44hiNK1.1low) NKT cells in the thymus (12) and exhibit a identification of ZBTB7B as a major switch factor represents an deficiency in peripheral NKT cells, which clearly distinguishes important step toward being able to control NKT cell function. this defect from that observed in Zbtb7b mutant NKT cells. Fur- The developmental and functional basis for CD4 and CD8 thermore, NK1.1 was not lower in GATA-3–deficient NKT cells, coreceptor expression by NKT cells has been a long-standing they were able to produce IFN-g, and they do not express CD8 puzzle in the field. Because NKT cell TCRs are CD1d re- (17). Thus, there appears to be at least two factors (GATA-3 and stricted, there is no obvious role for CD4 or CD8 in binding to MHC ZBTB7B) that are important for controlling CD4 expression by class II or MHC class I, respectively, nor is it clear how or why these NKT cells, and they work in a nonredundant manner. molecules are modulated during NKT cell development. Our data The precise mechanisms by which ZBTB7B regulates the de- using “helpless” Zbtb7b mutant mice sheds new light on this velopment of NKT cell subsets and how the Leu102Arg mutation problem, demonstrating that ZBTB7B is critical for development affects the function of the protein remain to be determined. It is 5248 DEVELOPMENT OF IL-17 PRODUCING NKT-CELLS DEPENDS ON ZBTB7B currently unclear if the L102R mutation is a null allele or has 2. Godfrey, D. I., and M. Kronenberg. 2004. Going both ways: immune regulation + via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379–1388. a dominant-negative effect, but as the number of RORgt , IL-17– 3. Godfrey, D. I., H. R. MacDonald, M. Kronenberg, M. J. Smyth, and L. Van Kaer. producing cells is intermediate in the heterozygous mice (Figs. 2004. NKT cells: what’s in a name? Nat. Rev. Immunol. 4: 231–237. 4A, 5B), this appears to support the concept that the mutation is 4. Coquet, J. M., S. Chakravarti, K. Kyparissoudis, F. W. McNab, L. A. Pitt, B. S. McKenzie, S. P. Berzins, M. J. Smyth, and D. I. Godfrey. 2008. Diverse having a null effect. This also fits well with the fact that the cytokine production by NKT cell subsets and identification of an IL-17- Leu102Arg mutation is in the dimerization domain (Supplemental producing CD4-NK1.1- NKT cell population. Proc. Natl. Acad. Sci. USA 105: Fig. 1) and very likely interferes with the dimerization of 11287–11292. 5. Gumperz, J. E., S. Miyake, T. Yamamura, and M. B. Brenner. 2002. Functionally ZBTB7B required for transcriptional activation, and this likely distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tet- results in a null allele in the homozygous state. Furthermore, ramer staining. J. Exp. Med. 195: 625–636. based on our observations from hpls/+ and hpls/hpls mice, the 6. Lee, P. T., K. Benlagha, L. Teyton, and A. Bendelac. 2002. Distinct func- tional lineages of human V(alpha)24 natural killer T cells. J. Exp. Med. 195: data suggest that full expression of this protein is required to 637–641. maintain normal numbers of CD4+ NKT cells because these are 7. Michel, M.-L., A. C. Keller, C. Paget, M. Fujio, F. Trottein, P. B. Savage, C.- H. Wong, E. Schneider, M. Dy, and M. C. Leite-de-Moraes. 2007. Identification diminished (but not absent) in the heterozygous mice. In contrast, of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway heterozygous mice showed no apparent defect in the percentage or neutrophilia. J. Exp. Med. 204: 995–1001. number of conventional CD4+ T cells in the periphery. It was only 8. Lee, P. T., A. Putnam, K. Benlagha, L. Teyton, P. A. Gottlieb, and A. Bendelac. 2002. Testing the NKT cell hypothesis of human IDDM pathogenesis. J. Clin. in the homozygous mutant mice that we found an absence of Invest. 110: 793–800. + + CD4 NKT cells and an abundance of CD8 NKT cells, sug- 9. van der Vliet, H. J., B. M. von Blomberg, N. Nishi, M. Reijm, A. E. Voskuyl, gesting that partial expression of ZBTB7B is still sufficient to A. A. van Bodegraven, C. H. Polman, T. Rustemeyer, P. Lips, A. J. van den Eertwegh, et al. 2001. Circulating V(alpha24+) Vbeta11+ NKT cell numbers are

+ Downloaded from prevent CD8 NKT cells. Whether this simply represents a dose decreased in a wide variety of diseases that are characterized by autoreactive effect acting at the same point in development or independent tissue damage. Clin. Immunol. 100: 144–148. effects at different stages in development is less clear. However, 10. Berzins, S. P., A. D. Cochrane, D. G. Pellicci, M. J. Smyth, and D. I. Godfrey. + 2005. Limited correlation between human thymus and blood NKT cell content the fact that CD8 NKT cells were far more abundant in spleen revealed by an ontogeny study of paired tissue samples. Eur. J. Immunol. 35: and liver compared with thymus supports the second scenario, 1399–1407. where an absence of functional ZBTB7B allows a delayed in- 11. Godfrey, D. I., S. Stankovic, and A. G. Baxter. 2010. Raising the NKT cell + family. Nat. Immunol. 11: 197–206. crease in CD8 NKT cells, which does not occur in the presence 12. Kim, P. J., S.-Y. Pai, M. Brigl, G. S. Besra, J. 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