Roquin Paralogs Differentially Regulate Functional NKT Cell Subsets Christoph Drees, J. Christoph Vahl, Sabrina Bortoluzzi, Klaus D. Heger, Julius C. Fischer, F. Thomas Wunderlich, This information is current as Christian Peschel and Marc Schmidt-Supprian of September 29, 2021. J Immunol published online 10 February 2017 http://www.jimmunol.org/content/early/2017/02/09/jimmun ol.1601732 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2017/02/09/jimmunol.160173 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 29, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 10, 2017, doi:10.4049/jimmunol.1601732 The Journal of Immunology

Roquin Paralogs Differentially Regulate Functional NKT Cell Subsets

Christoph Drees,*,1,2 J. Christoph Vahl,†,1,3 Sabrina Bortoluzzi,*,1 Klaus D. Heger,*,†,4 Julius C. Fischer,* F. Thomas Wunderlich,‡ Christian Peschel,* and Marc Schmidt-Supprian*,†

NKT cells represent a small subset of glycolipid-recognizing T cells that are heavily implicated in human allergic, autoimmune, and malignant diseases. In the thymus, precursor cells recognize self-glycolipids by virtue of their semi-invariant TCR, which triggers NKT cell lineage commitment and maturation. During their development, NKT cells are polarized into the NKT1, NKT2, and NKT17 subsets, defined through their cytokine-secretion patterns and the expression of key transcription factors. However, we have largely ignored how the differentiation into the NKT cell subsets is regulated. In this article, we describe the mRNA- binding Roquin-1 and -2 proteins as central regulators of murine NKT cell fate decisions. In the thymus, T cell–specific ablation of Downloaded from the Roquin paralogs leads to a dramatic expansion of NKT17 cells, whereas peripheral mature NKT cells are essentially absent. Roquin-1/2–deficient NKT17 cells show exaggerated lineage-specific expression of nearly all NKT17-defining proteins tested. We show through mixed bone marrow chimera experiments that NKT17 polarization is mediated through cell-intrinsic mechanisms early during NKT cell development. In contrast, the loss of peripheral NKT cells is due to cell-extrinsic factors. Surprisingly, Roquin paralog–deficient NKT cells are, in striking contrast to conventional T cells, compromised in their ability to secrete cytokines. Altogether, we show that Roquin paralogs regulate the development and function of NKT cell subsets in the thymus and http://www.jimmunol.org/ periphery. The Journal of Immunology, 2017, 198: 000–000.

atural killer T cells express an evolutionarily conserved Rare Va14-Ja18 (Va14i) TCRa-chain rearrangements in CD4 semi-invariant TCR and are characterized by an activated CD8 double-positive (DP) thymocytes lead, in conjunction with a N phenotype and rapid secretion of effector cytokines limited number of TCRb-chains, to the expression of a glycolipid- in response to innate and antigenic stimulation. In the mouse, recognizing TCR. The overwhelming majority of NKT cells ex- NKT cells represent 0.2–0.5% of lymphocytes in thymus, spleen, press this Va14i-containing semi-invariant TCR. Therefore, they ∼ and bone marrow and 30% in the liver, whereas in humans the are termed invariant NKT or Va14i-NKT cells, but for simplicity by guest on September 29, 2021 fractions are smaller (,0.1 and 1%, respectively) (1). Despite we will refer to them as NKT cells. Recognition of glycolipid Ags their rarity, NKT cell responses can drive inflammation or toler- presented by CD1d on DP thymocytes through the semi-invariant ance, thereby impacting a wide range of immune cells, such as TCR expressed by precursor cells (stage 0: CD24high, CD44low, dendritic cells, NK cells, and B and T cells (2). NKT cells protect NK1.12) triggers NKT cell development (10, 11). These TCR their host organisms from certain strains of bacteria, sustain an- signals induce massive proliferative expansion, downregulation of tiviral responses, and contribute to the suppression of certain types CD24 (stage 1), and expression of the key transcriptional regu- of cancer and immune diseases (1, 3–6). In contrast, NKT cells are lator of NKT cell maturation, promyelocytic leukemia zinc fin- involved in the pathophysiology of allergic responses, ulcerative ger (PLZF) (12–15). Thymic NKT cell maturation is indicated by colitis, and liver cancer (7–9). Therefore, NKT cell activation can subsequent upregulation of the memory marker CD44 (stage 2) have dramatically different outcomes, depending on diverse en- and expression of NK cell markers, most prominently NK1.1 vironmental factors. (stage 3) (16, 17). While passing through stages 1–3, NKT cells

*III. Medizinische Klinik fur€ Ha¨matologie und Onkologie, Klinikum Rechts der Isar, stipends from the Ernst Schering Foundation and the Boehringer Ingelheim Fonds, Technische Universita¨tMunchen,€ 81675 Munich, Germany; †Molecular Immunology respectively. and Signal Transduction Group, Max-Planck Institute of Biochemistry, 82152 Mar- C.D., J.C.V., S.B., and K.D.H. designed and performed experiments and analyzed tinsried, Germany; and ‡Obesity and Cancer Group, Max-Planck Institute for Meta- results; J.C.F. designed and performed experiments; F.T.W. provided essential re- bolism Research, 50931 Cologne, Germany agents and C.P. provided guidance; M.S.-S. designed the research and analyzed 1C.D., J.C.V., and S.B. contributed equally to this work and are considered first results; and C.D. and M.S.-S. wrote the article. authors. Address correspondence and reprint requests to Prof. Marc Schmidt-Supprian, De- 2Current address: Department of Internal Medicine III, Klinikum Grosshadern, partment of Hematology and Oncology, Klinikum rechts der Isar, Technische Uni- Munich, Germany. versita¨tMunchen,€ Ismaninger Strasse 22, 81675 Munchen,€ Germany. E-mail address: [email protected] 3Current address: Merck KGaA, Darmstadt, Germany. The online version of this article contains supplemental material. 4Current address: Physiological Chemistry, Genentech, Inc., South San Francisco, CA. Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; DN, double-negative; DP, double-positive; MFI, median fluorescence intensity; mLN, mesenteric lymph node; ORCIDs: 0000-0002-1683-8196 (J.C.V.); 0000-0002-6951-3416 (J.C.F.); 0000-0003- mTOR, mechanistic target of rapamycin; PLCg, phospholipase-Cg;pLN,peripheral 0547-2886 (C.P.). lymph node; PLZF, promyelocytic leukemia zinc finger; SLAM, signaling lymphocyte Received for publication October 6, 2016. Accepted for publication January 18, activation molecule; TFH, follicular Th; ThPOK, Th poxviruses and zinc finger and 2017. Kruppel€ family; TSC1, tuberous sclerosis 1.

This work was supported by the Deutsche Forschungsgemeinschaft through Grants Ó SCHM2440-3 and SFB 1054 A02 (to M.S.-S.). J.C.V. and K.D.H. received Ph.D. Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601732 2 ROQUIN PARALOGS POLARIZE NKT CELLS also differentiate into functional subsets termed NKT1 (PLZFlow, Samples were acquired on a FACSCanto II or LSRFortessa (BD) and T-bet+), NKT2 (PLZFhigh,GATA3+), and NKT17 (PLZFint, analyzed with FlowJo software. For gating on NKT cells, the following → RORgt+) cells, which are polarized toward the preferential pro- gating strategy was used: singlets LIVE/DEAD stain, gated on living cells → lymphocyte gate → TCRbint mCD1d-PBS57+ cells. NKT cell duction of cytokines reminiscent of the respective Th cell lineages stages and subsets were gated as indicated in the FACS plots. To ana- (18, 19). The signals and events that drive the differentiation of lyze the relative expression of extracellular and intracellular proteins, these NKT cell subsets are incompletely understood. median fluorescent intensities (MFIs) were calculated, and the mean MFI 2 + Roquin-1 and its mammalian paralog Roquin-2, encoded by of mCD1d-PBS57 tetramer TCRb T cells of the respective control mice was set to 1. rc3h1 and rc3h2 respectively, are mRNA-binding proteins that repress immune reactions by redundant and unique posttran- Isolation of lamina propria and lung lymphocytes scriptional regulation of genes, including costimulatory molecules For isolation of lamina propria lymphocytes, Peyer’s patches and fat tissue and proinflammatory cytokines (20–24). Thereby, these proteins were removed, and intestines were flushed with ice-cold PBS. Intestines act as safeguards against autoimmunity by preventing the spon- were opened longitudinally and cut into 1–1.5-cm pieces. After vigorously vortexing in PBS, samples were incubated two times for 15–20 min at taneous generation of follicular Th (TFH) cells, IL-17–producing Th17 cells, and IFN-g–producing short-lived effector CD8 T cells 37˚C in HBSS buffer containing 5% FCS and 5 mM EDTA, 1 mM DTT, and 10 mM HEPES. Then, cells were washed in PBS, and intestines were (20, 22, 25). However, their role in the development of agonist- digested for 45 min at 37˚C in PBS+Ca/+Mg containing 5% FCS, 1 mg/ml selected T cell subsets, such as NKT cells, is unknown. Collagenase, Type 2 (CellSystems), and 0.1 mg/ml DNase I (Roche). In this article, we report that conditional ablation of both Roquin Lymphocytes were then purified by a 40/80% Percoll (Biochrom) gradient. paralogs in T cells essentially prevents the generation of mature To prepare single-cell suspensions, lungs were digested using RPMI 1640 containing 0.02 mg/ml Liberase TM and 10 U DNase (both from NKT cells in the thymus, with the exception of NKT17 cells, whose Roche Diagnostics). Downloaded from production is dramatically increased by cell-intrinsic mechanisms. Roquin-1/2–deficient NKT cells express high amounts of NKT17- Bone marrow chimeras associated markers but display a hyporesponsive phenotype, as B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL-congenic); C57BL/6 heterozygous shown by impaired cytokine production. In addition, peripheral recipient mice (CD45.1/2) were lethally irradiated with 2 3 5.5 Gy (4 h Roquin-1/2–deficient NKT cells are essentially absent, mainly due apart) and injected i.v. with 4–5 3 106 bone marrow cells in a B6.SJL (CD45.1)/C57BL/6 (CD45.2) ratio of 1:1. Before transplantation, bone to cell–extrinsic mechanisms. http://www.jimmunol.org/ marrow was depleted of T cells using CD90.2 MicroBeads (Miltenyi Biotec). Two to three days before until 2 wk after transplantation recipient Materials and Methods mice were treated with Borgal 24% (Virbac Animal Health) in drinking Mice water at a dose of 0.1 ml/kg body weight/d. Mice were analyzed 7–9 wk after transplantation. CD4Cre (26), Rc3h1F/F (24), Rc3h2F/F (22), Va14iStopF (27), IL6Ra2/2, and IL6RaF/F (28) mice were kept on a C57BL/6 genetic background. Statistical analysis Mice were housed in specific pathogen–free animal facilities at the Max- Planck Institute of Biochemistry and the Technische Universita¨tMunchen.€ Statistical analysis was performed by GraphPad prism version 6. The Unless indicated otherwise, age-matched animals were analyzed at 6–12 wk p values were calculated as indicated in the figure legends. of age. CD4Cre Rc3h1-2F/F mice were analyzed before any visible signs of by guest on September 29, 2021 pathology. All experiments were performed in accordance with German Results Federal Animal Protection Laws and approved by the Regierung of Ober- bayern. In most experiments, Rc3h1F/F Rc3h2F/F, Rc3h1F/F Rc3h2F/+, and/or Ablation of Roquin proteins leads to a reduction in thymic CD4Cre mice were used as controls. Controls were pooled, because one NKT cells and a loss of peripheral NKT cells copy of the CD4Cre transgene does not significantly affect thymocyte numbers or splenic NKT cell numbers (29). To assess the role of Roquin proteins in NKT cells, we analyzed mice with T cell–specific ablation of one or both of these proteins. Flow cytometry We used CD4Cre for this purpose, because it efficiently recom- F/F F/F Single-cell suspensions were stained using the following commercial Abs bines conditional alleles, including Rc3h1 (24) and Rc3h2 and kits: Bcl-6 (K112-91), B220 (RA3-6B2), CCR-6 (29-2L17), CD4 (22), in DP thymocytes. CD4Cre-mediated protein ablation is (GK1-5, RM4-5), CD8a (53-6.7), CD11b (M1/70), CD44 (IM7), CD45.1 complete in NKT cell precursors (16, 17, 30, 31), which represent (A20), CD45.2 (104), CD62L (MEL-14), CD90.2 (53-2.1), CD127 (A7R34), CD138 (281-2), c-MAF (sym-0F1), CXCR5 (2G8), Egr2 older DP thymocytes because of the late timing of the Va14-Ja18 (erongr2), Foxp3 (FJK-16s), GATA3 (TWAJ), Gr-1 (RB6-8C5), IL-17 rearrangement (16, 17, 31, 32). Knockout of Roquin-1 and coa- (eBio17B7), IL-4, (11B11), IL-6Ra (D7715A7), INF-g (XMG1.2), Ly- blation of Roquin-1/2 led to a 2-fold reduction in NKT cell pro- 6C (HK1.4), Neuropilin-1 (JE12), NK1.1 (PK136), PD-1 (J43), PLZF portions and numbers in the thymus (Fig. 1A, 1B). Analysis of (9E12), RORgt (AFKJS-9, Q31-387), TCRb (H57-597), T-bet (4B10), and thymic developmental stages 1–3 revealed normal numbers at ThPOK (2POK). For intracellular transcription factor staining, cells were fixed and permeabilized with a Foxp3 Transcription Factor Staining Buffer stage 1, gain of numbers at stage 2, and significant loss of numbers F/F Set (eBioscience). For intracellular cytokine staining, cells were fixed and at stage 3 in NKT cells in CD4Cre Rc3h1 mice compared with permeabilized with a Cytofix/Cytoperm kit (BD) or a Foxp3 Transcription control mice; this was strongly exacerbated when Roquin-2 was Factor Staining Buffer Set (eBioscience). For analysis of intracellular coablated (Fig. 1A, 1C). Accordingly, the absolute numbers of phosphorylated proteins, single-cell suspensions were prepared using stage 3 NKT cells were reduced from an average of 32 3 104 cells serum-free buffer. Cells were processed using a Transcription Factor 4 F/F 4 Phospho Buffer Set or fixed with Phosflow Lyse/Fix Buffer and per- in controls to 16 3 10 in CD4Cre Rc3h1 mice and 1 3 10 in meabilized with Perm Buffer III (all from BD). Cells were stained over- CD4Cre Rc3h1-2F/F mice (Fig. 1C). night using commercially available mAbs against p-STAT3 (pS727), Moreover, loss of Roquin proteins also affected NKT cells in p-mTOR (MRRBY), p-4EBP1 (236B4) and p-AKT (S473) (M89-61), peripheral lymphoid organs. In the spleens of CD4Cre Rc3h1F/F p-ZAP70 (n3kobu5), and phosphorylated phospholipase-Cg (PLCg) (K86-689.37) (all from BD). For detection of apoptotic cells, cells were mice, NKT cell proportions and absolute numbers were reduced stained with Annexin V and 7-aminoactinomycin D (7-AAD) using an by .50%, primarily due to a reduction in stage 3 NKT cells Annexin V detection kit (eBioscience). mCD1d PBS-57 tetramers were (Fig. 1D, 1E, Supplemental Fig. 1A). Strikingly, NKT cells provided by the National Institutes of Health Tetramer Core Facility. For were virtually absent in the spleens of CD4Cre Rc3h1-2F/F mice enrichment of thymic NKT cells, CD8+ thymocytes were depleted using CD8a MicroBeads (Miltenyi Biotec). For intracellular cytokine staining, (Fig. 1D, 1E). A similar picture was observed in the liver, pe- cells were stimulated for 4 h with 100 ng/ml PMA (Sigma) and 1000 ng/ml ripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), ionomycin (Calbiochem) together with 2 mM monensin (eBioscience). and lung (Fig. 1F, Supplemental Fig. 1B). Immune cells were The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Ablation of Roquin paralogs leads to an expansion of stage 2 NKT cells and reduction of NKT cells in the periphery. (A) Percentage of thymic mCD1d- PBS57 tetramer+ NKT cells and NKT cell stages in control, CD4Cre Rc3h1F/F,andCD4Cre Rc3h1-2F/F mice. Numbers in representative plots indicate mean percentage 6 SD calculated from at least eight mice per genotype pooled from at least three independent experiments. (B) Absolute cell numbers (3104)ofthymic NKT cells of the indicated genotypes. Each data point represents one mouse, andthebarsindicatethemeancellnumber,whichisalsodepictedbelowthegraph. Graph shows pooled data from at least three independent experiments. **p , 0.01, ****p , 0.0001, one-way ANOVA. (C) Absolute cell numbers (3104)ofthymicNKTcell stages of the indicated genotypes. Bars show the mean numbers. Each data point represents one mouse of the indicated genotype of at least three independent ex- periments. *p , 0.05, multiple t tests. (D) Spleens of control, CD4Cre Rc3h1F/F,andCD4Cre Rc3h1-2F/F mice were analyzed for the presence of NKT cells. Shown are representative contour plots of each genotype gated on total lymphocytes. Numbers indicate mean percentage 6 SD calculated from a total of at least seven mice per genotype pooled from at least two independent experiments. (E) Total cell numbers (3104) of NKT cells in the spleens of control, CD4Cre Rc3h1F/F,andCD4Cre Rc3h1-2F/F mice. Bars represent the mean cell number (also depicted below the graph), with each data point representing one analyzed mouse pooled from at least two independent experiments. *p , 0.05, **p , 0.01, ****p , 0.0001, one-way ANOVA. (F) Total cell numbers (3104) of lymphocytes and NKT cells in the liver, pLNs, and mLNs of control and CD4Cre Rc3h1-2F/F mice. Bars indicate the mean cell number, with each data point representing one mouse. Data are pooled from at least six mice per genotype from at least two independent experiments. *p , 0.05, **p , 0.005, unpaired t test. ns, not significant. 4 ROQUIN PARALOGS POLARIZE NKT CELLS dramatically expanded in liver and most prominently in pLNs, but Rc3h1-2F/F mice, expression of Cre leads to recombination of all not in mLNs, of CD4Cre Rc3h1-2F/F mice compared with control conditional alleles, ultimately causing the inactivation of the mice. Therefore, absolute NKT cell numbers were reduced in liver Roquin-1/2 alleles and induction of the Va14i TCRa-chain. and mLNs but not pLNs (Fig. 1F). In contrast to Roquin-1, indi- Va14i TCR expression occurs within 6 h after Cre expression vidual loss of Roquin-2 has no discernable effects on NKT cell (S. Bortoluzzi, C. Drees, and M. Schmidt-Supprian, unpublished development and numbers (Supplemental Fig. 1C, 1D). observations), whereas it takes considerably longer before the cells Taken together, ablation of Roquin proteins leads to a dose- are functionally depleted of Roquin-1/2 proteins (S. Bortoluzzi, dependent decrease in thymic NKT cell generation, with a clear C. Drees, and M. Schmidt-Supprian, unpublished observations). maturation block between stages 2 and 3. In the periphery, Furthermore, it is very likely that a cell has activated the NKT cells were only barely detectable in CD4Cre Rc3h1-2F/F Va14iStopF allele before it has inactivated all four Roquin alleles. mice. Therefore, in contrast to the situation in CD4Cre Rc3h1-2F/F mice, in CD4Cre Va14iStopF Rc3h1-2F/F mice a newly expressed Roquin proteins restrict NKT17 cell differentiation Va14i TCR initially signals to a cell that still contains some Given that most NKT17 cells are phenotypically stage 2, we de- Roquin proteins, which are then lost during NKT cell develop- termined the subset composition (33). We found increased pro- ment. Va14i TCR expression restored the development of portions of NKT17 cells in CD4Cre Rc3h1F/F mice, whereas most NKT cells of all stages in CD4Cre Va14iStopF Rc3h1-2F/F mice, of the NKT cells in CD4Cre Rc3h1-2F/F mice were NKT17 although the overall numbers were still significantly reduced (Fig. 2A). Loss of Roquin-2 alone had no effect on NKT subset compared with controls (Fig. 3A, 3B). In Va14i knock-in mice, composition (Supplemental Fig. 1E). Importantly, the absolute NKT cell development is strongly biased toward cells early in Downloaded from numbers of NKT17 cells were increased .10-fold, from an av- their development (Fig. 3B), which is also reflected in the ex- erage of 0.5 3 104 in controls to 6.5 3 104 cells in CD4Cre pression profiles of RORgt and PLZF (Supplemental Fig. 2A). Rc3h1-2F/F mice. Therefore, we conclude that the increased pro- Within the mCD1d-PBS57 tetramer+ NKT cells, we defined the portions are due to strongly increased NKT17 cell production in earliest RORgt+ PLZF2 CD442 CD24+ progenitor cells as the absence of Roquin-mediated regulation and not merely a Va14i-DP, because they essentially represent DP thymocytes

consequence of a decreased NKT1 population (Fig. 2B). At this expressing a Va14i-NKT cell TCR (Fig. 3D, 3E, Supplemental http://www.jimmunol.org/ point, because we observed more pronounced changes in the ab- Fig. 2A). We termed the putative next RORgt2 PLZF2 NK1.12 sence of both Roquin proteins, we focused on the analysis of developmental stage as pre-NKT, because it consists of various thymic NKT cells from CD4Cre Rc3h1-2F/F mice in further NKT cell precursor populations (Supplemental Fig. 2A). Pro- experiments. portions and absolute cell numbers indicated a slight block in the ICOS is the first described target of Roquin’s posttranscriptional Va14i-DP to pre-NKT transition upon ablation of Roquin-1/2 activities, but ICOS is also differentially expressed among NKT cell (Fig.3C).ThenumbersofNKT1(RORgt2, NK1.1+, PLZFint) subsets. Therefore, we compared ICOS MFIs on NKT1, NKT2, and and NKT17 (RORgt+,CD44+,CD242)cellsweresimilarinboth NKT17 cells. In controls, the highest ICOS expression was found genotypes, whereas loss of Roquin caused a decrease in NKT2 on NKT17 and NKT2 cells, whereas NKT1 cells expressed similar (RORgt2, PLZFhigh,CD242) cells (Fig. 3C, Supplemental Fig. by guest on September 29, 2021 amounts as TCRb+ tetramer2 thymocytes (Fig. 2C). Loss of both 2A). Further characterization of the early progenitor stages and Roquin paralogs enhanced ICOS expression to a similar extent in all of the functional subsets with T-bet and GATA3 confirmed these subsets (Fig. 2C), indicating that the differential ICOS expression in results (Fig. 3D–G). Mature Roquin-1/2–deficient NKT cells of NKT cell subsets occurs largely independently of Roquin-mediated all subsets and stages also were detected in the spleen, although regulation. the numbers were reduced compared with controls (Fig. 4). Most NKT17 cells are characterized by enhanced expression of the diminished were PLZFhigh (NKT2) cells, whereas NKT17 cell chemokine receptor CCR-6, the integrin aEb7 (CD103), syndecan-1 numbers were not significantly altered (Fig. 4). (CD138), and neuropilin-1 (Nrp-1) (Fig. 2D) (34–36). Unlike In conclusion, we show that expression of a knock-in Va14i controls, Roquin-1/2–deficient NKT17 cells were uniformly posi- NKT cell TCR, concomitant with ablation of Roquin-1/2, largely tive for all of these markers (Fig. 2D). Interestingly, a fraction of overcomes the block in thymic NKT cell development, permits the Roquin-1/2–deficient NKT1 and NKT2 cells expressed CD138, the accumulation of Roquin-deficient mature peripheral NKT cells, surface protein most faithfully representing NKT17 lineage com- and strongly reduces the bias toward NKT17 cell production in mitment. This could indicate a bias toward NKT17 differentiation in CD4Cre Va14iStopF Rc3h1-2F/F mice. these cells. In summary, we demonstrate that Roquin proteins prevent po- Roquin paralogs cell intrinsically drive NKT17 differentiation, larization into the NKT17 lineage. and the loss of peripheral NKT cells is also due to cell-extrinsic factors a Expression of a knock-in V 14i TCR facilitates development The results derived from experiments with the Va14i TCR indi- and peripheral accumulation of all NKT cell subsets in StopF F/F cated that Roquin proteins play decisive roles during the earliest CD4Cre Va14i Rc3h1-2 mice stages of NKT cell development. DP thymocytes are critically Transgenic expression of a Va14i TCR has been used frequently important for positive selection of developing NKT cells by pre- to rescue defects in early NKT cell development. The presence senting Ag and through stimulation via homotypic interactions of normal numbers of stage 1 NKT cell precursors in CD4Cre between signaling lymphocytic activation molecule (SLAM) Rc3h1-2F/F mice essentially excludes defects in TCRa rear- family members (16, 37). However, the expression of CD1d, rangements or thymocyte survival, which are commonly overcome CD150 (SLAMF1), and Ly108 (SLAMF6), which are required for in such experiments. We recently generated the Va14iStopF allele, successful NKT cell development (37), were not significantly al- a novel type of Va14i transgene that expresses a Va14i TCR from tered by the lack of Roquin paralogs in DP thymocytes (Fig. 5A), within the endogenous TCRa gene locus upon Cre-mediated re- arguing against an indirect role for these cells. To directly dis- combination. In combination with CD4Cre, this leads to massive tinguish between cell-intrinsic or cell-extrinsic mechanisms, we NKT cell overproduction (Fig. 3A) (27). In CD4Cre Va14iStopF created mixed bone marrow chimeras. We adoptively transferred The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Ablation of Roquin promotes polarization into the NKT17 lineage. (A) Percentage of NKT1, NKT2, and NKT17 cells in the thymi of control, CD4Cre Rc3h1F/F, and CD4Cre Rc3h1-2F/F mice. Numbers in the representative contour plots indicate mean percentage 6 SD calculated from at least eight mice per genotype of at least three independent experiments. Cells were acquired after MACS depletion of CD8+ thymocytes. (B) Absolute cell numbers (3104) of thymic NKT cell subsets of the indicated genotypes. Bars show the mean cell number, with each data point representing one mouse. Shown data are pooled from at least three independent experiments. (C) ICOS MFI on thymic tetramer2 TCRb+ thymocytes and NKT1, NKT2, and NKT17 cells of the indicated genotypes. Bars show mean MFI, and each data point represents one mouse. MFI was normalized to the mean MFI of control TCRb+ tetramer2 thymocytes, which was set to 1. Shown data are pooled from at least three independent experiments. (D) CCR-6, CD103, CD138, and neuropilin-1 ex- pression on thymic NKT1, NKT2, and NKT17 cells of control and CD4Cre Rc3h1-2F/F mice. Numbers in representative contour plots show the mean percentage 6 SD calculated from at least three mice per marker pooled from at least two independent experiments. Thymi in (A), (C), and (D) were depleted of CD8 by MACS before acquisition. *p , 0.05, multiple t tests using the Holm–Sidak method. 6 ROQUIN PARALOGS POLARIZE NKT CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Transgenic expression of the Va14i TCR at the DP stage enables the development of diverse thymic NKT cell subsets in CD4Cre Rc3h1-2F/F mice. Representative contour plots show the mean percentage 6 SD, and bar charts show the total cell numbers (3104) of thymic NKT cells (A) and NKT cell stages (B) in mice of the indicated genotype. (C) Bar charts show the percentage and absolute cell numbers (3104)ofthymicNKTcellsubsetsinCD4Cre Va14iStopF and CD4Cre Va14iStopF Rc3h1-2F/F mice. NKT cell subsets were defined as RORgt+,PLZF2,CD24+,CD442 (Va14i-DP cells), RORgt2, non-NKT2 and non-NKT1 cells (pre-NKT cells), RORgt2,NK1.1+,PLZFlow (NKT1 cells), RORgt2,PLZFhigh,CD242 (NKT2 cells), and RORgt+,PLZFint,CD242,CD44+ (NKT17 cells). Bars in (A–C) indicate the mean cell number, with each data point representing one analyzed mouse. Data were pooled from at least three independent experiments. *p , 0.05, multiple t tests using the Holm–Sidak method. (D) Representative contour plots show the gating strategy to define thymic Va14i-DP (RORgt+,PLZF2, CD24+), pre-NKT (RORgt2, non-NKT2 and non-NKT1 cells), NKT1 (RORgt2,T-bet+,PLZFlow), NKT2 (RORgt2,GATA3+,PLZFhigh), and NKT17 (RORgt+, PLZFint,CD242) cells in CD4Cre Va14iStopF mice. (E) Contour plots show expression of CD44 and CD24 (upper panels) and CD4 and CD8 (lower panels) in thymic Va14i-DP and pre-NKT cells. Plots are representative of three CD4Cre Va14iStopF mice of one experiment. Bar charts show the percentage (F)andabsolute cell numbers (G)ofthymicVa14i-DP, pre-NKT, NKT1, NKT2, and NKT17 cells in CD4Cre Va14iStopF and CD4Cre Va14iStopF Rc3h1-2F/F mice, gated as indicated in (D). Bars show the mean percentage (F) and mean cell number 3 104 (G), with each data point representing one individual mouse of one experiment. *p , 0.05, multiple t tests (G) using the Holm–Sidak method (F). ns, not significant. The Journal of Immunology 7

FIGURE 4. NKT cells in the spleens of CD4Cre Va14iStopF and CD4Cre Va14iStopF Rc3h1-2F/F mice. (A) Representative contour plots show the mean Downloaded from percentage 6 SD of splenic NKT cells (left panel), NKT cell stages 1–3 (middle panel), and NKT1, NKT2, and NKT17 cells (right panel) in CD4Cre Va14iStopF mice compared with CD4Cre Va14iStopF Rc3h1-2F/F mice. (B) Bar charts show the mean cell numbers of splenic NKT cells (left panel), NKT stages 1–3 (middle panel), and NKT1, NKT2, and NKT17 cells (right panel), gated as shown in (A), and isolated from spleens of the indicated genotypes. Each symbol represents one analyzed mouse, and all shown data were pooled from three independent experiments. *p , 0.05, multiple t tests using the Holm–Sidak method. ns, not significant. http://www.jimmunol.org/ T cell–depleted CD45.1 B6.SJL-congenic bone marrow, mixed Thus, our results demonstrate that the massive NKT17 cell with an equal amount of CD45.2 control or CD45.2 CD4Cre production in CD4Cre Rc3h1-2F/F mice is mediated through cell- Rc3h1-2F/F T cell–depleted bone marrow, into lethally irradiated intrinsic mechanisms, whereas the reduction in mature peripheral CD45.1/2 DP mice (Fig. 5B, Supplemental Fig. 2B). Thymi and NKT cells is largely caused by cell-extrinsic mechanisms. spleens of the resulting chimeras were analyzed 7–9 wk after transplantation. Even after lethal irradiation, 20–30% of thymic Roquin-1/2–deficient NKT cells are characterized by elevated NKT cells in the resulting chimeras were derived from hosts, and c-Maf but reduced levels of Egr2, PLZF, and phosphorylated similar proportions of radio-resistant CD45.1/2 host T and STAT3 NKT cells were found in the spleens (Supplemental Fig. 2C). Having demonstrated the cell-intrinsic nature of the NKT17 bias by guest on September 29, 2021 Further, to account for the engraftment variations that are typical of Roquin-1/2–deficient NKT cells, we embarked on further mo- of these experiments, we normalized the CD45.1/CD45.2 ratios to lecular characterization. Compared with controls, NKT17 cells those of double-negative (DN) thymocytes and B cells, which are lacking Roquin paralogs express higher amounts of TCR on their Roquin proficient. This analysis revealed that Roquin-1/2–deficient surface, whereas NKT1 cells express lower levels (Fig. 7A). NKT cells develop in normal proportions from their precursors, Conventional CD4 T cells upregulate Ly-6C upon deprivation of similar to other thymocyte subsets and similar to CD45.1 compet- TCR-mediated self-recognition (39). Within the NKT lineage, itor and CD45.2 control NKT cells. Strikingly, Roquin-1/2–deficient NKT1 cells had the largest proportion of Ly-6C+ cells, whereas NKT cells could also be detected at nearly normal proportions in only a few NKT2 and essentially no NKT17 cells were putatively the spleen, in striking contrast to the situation in CD4Cre Rc3h1-2F/F self-deprived, which inversely correlated with TCRb expression. mice (Figs. 1D, 1E, 5C, Supplemental Fig. 2C). The proportions Loss of Roquin-1/2 decreased the percentage of Ly-6C+ cells in all of Roquin-1/2–deficient CD45.2 thymic NKT17 cells were strongly NKT subsets, although the differences were not dramatic and significantly increased, in contrast to the CD45.1 competitor (Fig. 7B). Next, we assessed the expression of the TCR-induced and CD45.1 and CD45.2 cells in the control chimeras (Fig. 5D). transcription factor Egr2, which was also shown to induce PLZF Roquin-1/2–deficient NKT cells of all subsets could be detected in by binding to its promoter in immature NKT cells (40). Loss of the spleen, although NKT17 cells remained dominant (Fig. 5C, Roquin-1/2 led to a significant decrease in Egr2 levels in TCR+ Supplemental Fig. 2C, 2D). Furthermore, we found that elevated thymocytes and all NKT subsets, including NKT17 cells, which ICOS expression and the spontaneous differentiation of memory/ had the highest TCR surface levels (Fig. 7C). In Roquin-1/2– effector-like T cells and TFH cells of the Roquin-1/2–deficient deficient NKT17 cells, decreased Egr2 expression was accompa- T lineage are cell intrinsic (Supplemental Fig. 3), similar to the san/ nied by a 50% reduction in PLZF protein (Fig. 7C), but PLZF san T lineage (38). expression was unchanged in the other subsets. PLZF was shown To test whether peripheral NKT cells are initially generated but to regulate the expression of c-Maf (41), a transcriptional regu- lost over time in CD4Cre Rc3h1-2F/F mice, we analyzed NKT cell lator of cytokine production that is highly expressed in Th17 cells development in very young (16–20-d-old) mice. NKT cell num- (42) and contributes to Th17 differentiation through the induction bers were reduced significantly in the thymi and were nearly ab- of RORgt (43). Interestingly, we found significantly increased sent in the spleen of young CD4Cre Rc3h1-2F/F mice compared c-Maf expression in Roquin-1/2–deficient NKT2 and NKT17 cells, with control mice (Fig. 6A, 6B). At this age, NKT2 cells represent independent of PLZF levels (Fig. 7C). To assess the direct con- the largest subset in thymi of control mice, whereas in the absence tribution of TCR signals to these changes, we analyzed phos- of the Roquin paralogs, NKT17 cells predominate, and very few phorylation of the TCR proximal adapter molecule ZAP70 and NKT1/2 cells are generated (Fig. 6C), similar to the situation in the further downstream acting PLCg (Fig. 7C). In Roquin-1/2– older mice (Fig. 2B). deficient NKT17 cells, there was a trend toward diminished 8 ROQUIN PARALOGS POLARIZE NKT CELLS Downloaded from http://www.jimmunol.org/

FIGURE 5. The expansion of NKT17 cells after Roquin ablation is regulated in a cell-intrinsic manner; however, NKT cell-extrinsic mechanisms contribute to the reduction in peripheral NKT cells. (A) Representative line graphs showing the expression of Ly108 (SLAMF6), CD150 (SLAMF1), and CD1d on DP thymocytes of the indicated genotypes normalized to TCRb+ tetramer2 cells of control mice. Data represent the mean normalized MFI 6 SD by guest on September 29, 2021 of four mice pooled from three independent experiments. For normalization, MFI of TCRb+ tetramer2 cells was set to 1. (B) Experimental set-up of the bone marrow chimera experiment. CD45.1/2 DP recipient mice were transplanted with a 1:1 mixture of CD45.1 B6.SJL bone marrow together with CD45.2 control or CD45.2 CD4Cre Rc3h1-2F/F bone marrow. Bone marrow was depleted of T cells by MACS using CD90.2 beads. Mice were sacrificed 7–9 wk after transplantation, and NKT cells in thymi and spleens were analyzed. (C) The indicated thymic (upper panels) and splenic (lower panels) immune cell populations of both groups were analyzed for the expression of CD45.1 and CD45.2. Data represent mean percentage 6 SD of seven experimental and four control mice from one of two independent experiments and are normalized to the engraftment of DN thymocytes (thymus) or B cells (spleen). (D) Representative dot plots show the distribution of NKT1, NKT2, and NKT17 cells in the thymi of the indicated bone marrow chimeras. Cells were acquired after MACS depletion of CD8+ thymocytes. Numbers indicate mean percentages 6 SD calculated from four controls and seven experimental mice of one of two independent experiments. *p , 0.05, multiple t tests using the Holm–Sidak method. ns, not significant. intracellular TCR signals, but it was not statistically significant. with the exception of NKT17 cells, in which they were reduced Altogether, our analyses indicated that TCR signaling is not (Fig. 7D), possibly reflecting their strong polarization into this significantly impacted by the loss of Roquin-1/2 proteins. IL-6, lineage. together with TCR signals, strongly induces c-Maf expression To address the loss of NKT1 and NKT2 cells we monitored cell via STAT3 (44). However, in accordance with previous data (45), death, which revealed significantly increased apoptosis in Roquin- we did not detect significant expression of the STAT3-activating 1/2–deficient and control NKT cells at stage 2 (Fig. 7E) harboring IL-6R on bulk NKT cells. Complete or T cell–specific ablation .90% of NKT17 cells. This indicates that Roquin-1/2 do not of the IL-6Ra–chain did not affect NKT cell development and directly influence cell death but that their absence directs NKT cell subset differentiation in the thymus, but it led to increased differentiation into a proapoptotic compartment. NKT cells (47), splenic NKT cell numbers (Supplemental Fig. 4). Nevertheless, and especially NKT17 cells, depend on IL-7 for their generation, STAT3 phosphorylation was increased in control NKT2 and homeostasis, and survival (48). Accordingly, control NKT17 cells NKT17 cells compared with NKT1 cells (Fig. 7C), indicat- expressed three times more IL-7R a-chain (CD127) than did NKT1 ing that another receptor might activate STAT3. However, this and NKT2 cells. Loss of Roquin-1/2 led to a selective increase in receptor or downstream signals do not appear to be regulated CD127 expression in NKT17 cells (Fig. 7F), suggesting that by Roquin proteins, because Roquin-1/2–deficient NKT1 and decreased IL-7 signaling does not underlie their enhanced pro- NKT17 cells showed slightly reduced STAT3 phosphorylation pensity to die. compared with controls (Fig. 7C). Fate decisions between NKT1 Together, our data indicate that Egr2 signals and STAT3- and NKT17 cells are controlled by the transcription factor Th activating signals are diminished in Roquin-1/2–deficient NKT cells. poxviruses and zinc finger and Kruppel€ family (ThPOK), whose Mechanisms, including undefined signals that enhance c-Maf ex- expression is strongly reduced in NKT17 cells (34, 46). ThPOK pression, strongly drive them into the NKT17 lineage, where they levels were elevated in the absence of Roquin-1/2 in all subsets, are more prone to die. The Journal of Immunology 9

FIGURE 6. NKT cell development in young CD4Cre Rc3h1-2F/F mice. Bar charts represent the mean percentages (left panel) and absolute numbers (right panel) of thymic (A) and splenic (B) NKT cells isolated from 16–20-d-old CD4Cre Rc3h1-2F/F and control mice. Each data point represents one analyzed mouse; data were pooled from at least two independent experiments. **p , 0.005, ***p , 0.0005, unpaired t test. (C) Thymic and NKT cell subsets in CD4Cre Rc3h1-2F/F and control animals. Mean percentages (left panel) and mean cell numbers (right panel) of the indi- cated NKT cell subsets. Each data point repre- sents one analyzed mouse; data were pooled from at least two independent experiments. *p , 0.05, multiple t tests using the Holm–Sidak method. Downloaded from

Roquin paralog–deficient NKT cells are compromised in their PLZF regulation, because enhanced PLZF levels, as seen in lethal-7 cytokine-secretion abilities miRNA-deficiency, shift the balance from NKT1 to NKT2/17 The differentiation of NKT cell subsets and cytokine production (52). Ablation or loss-of-function mutations in ThPOK result also depend on signaling through the mechanistic target of rapa- in the production of CD8 and DN NKT cells that are charac- http://www.jimmunol.org/ mycin (mTOR) protein complexes (49), and ablation of the neg- terized by low NK1.1 expression (34, 53, 54). This goes hand in ative regulator tuberous sclerosis 1 (TSC1) leads to phenotypes hand with a dramatic polarization toward NKT17 differentiation, that strikingly resemble the phenotypes caused by loss of Roquin- which is also observed, although to a much lesser extent, in hap- 1/2 (50). However, we did not find evidence for increased mTOR loinsufficiency (34, 46). The elevated ThPOK levels in Roquin- signaling in Roquin-1/2–deficient NKT cells compared with 1/2–deficient thymocytes and NKT1/2 cells indicate that lower controls. There was no difference in the phosphorylation of mTOR ThPOK levels in NKT17 cells are the consequence, rather than or downstream proteins, such as 4-EBP1 (mTORC1) and AKT the cause, of exaggerated NKT17 differentiation in the absence (mTORC2) (Fig. 8A). Roquin deficiency controls the differentia- of Roquin-1/2. by guest on September 29, 2021 tion of proinflammatory T lymphocytes, as well as their effector A significant reduction in ThPOK mRNA was also detected functions (Fig. 8D) (25, 51). To our surprise, PMA/ionomycin in NKT cells deficient for the mTOR negative-regulator TSC1 (50), stimulation induced significantly lower proportions of cytokine- which show similar phenotypes, including a dramatic NKT17 producing cells in all Roquin-1/2–deficient NKT cell subsets bias and a reduction in mature thymic and peripheral NKT cells. compared with controls (Fig. 8B). Furthermore, cytokine pro- This phenotype was attributed and functionally linked to elevated duction per cell appeared to be lower in Roquin-1/2–deficient mTORC1 activity, reduced expression of T-bet, and ICOS-dependent NKT17 cells (Fig. 8C), whereas conventional effector T cells pro- signals (50). In our studies, we did not detect significant differ- duced significantly more proinflammatory cytokines after Roquin ences in mTORC1 or mTORC2 pathway activation in Roquin-1/ ablation (Fig. 8D). 2–deficient NKT17 cells compared with control cells. Further- Therefore, in contrast to the increased expression of NKT17- more, at least in the CD8 T cell lineage, loss of Roquin activity specific markers, Roquin-1/2–deficient NKT cells showed a strongly enhances the acquisition of an effector state that is hyporesponsive phenotype that was characterized by overall de- characterized by high T-bet expression (24, 51). In TSC1-deficient fective cytokine production. NKT cells, reduced Tbet levels might facilitate the transcrip- tional acquisition of high ICOS expression, which contributes to NKT17 polarization (50). A similar outcome with respect to ele- Discussion vated ICOS and NKT17 differentiation was independently achieved During their development in the thymus, NKT cells pass sequential through enhanced translation of ICOS mRNA in a Roquin-1/2– stages and mature into functionally different subsets. In this article, deficient situation. we show that the RNA-binding Roquin-1 and -2 proteins prevent ICOS, IL-6 in conjunction with TCR-induced Ca2+ signals (44), excessive differentiation into the NKT17 lineage. The numbers of TGF-b (55), and IL-27 (56) are all implicated in the regulation of NKT17 cells deficient for the Roquin paralogs exceeded their c-Maf expression. c-Maf is characteristically overexpressed in controls by .10-fold and showed strongly enhanced lineage Th17 cells, TFH cells (42), and, as we show in this article, NKT17 commitment. Mixed bone marrow chimeras demonstrated that cells. Normal TCR signal strength in NKT cells argues against a NKT17 polarization is cell intrinsic, and conditional expression of role for TCR signaling in enhancing c-Maf expression in the ab- aVa14i TCR, concomitantly with Roquin-1/2 ablation, suggested sence of Roquin-1/2. Because we and other investigators (45) that signals early during NKT cell development play decisive showed that IL-6R signals are dispensable for NKT17 develop- roles. ment, it seems more likely that cytokines, such as IL-27, trigger It remains unclear when and how the NKT17 fate is initiated c-Maf expression in developing NKT17 cells. IL-27 also induces during NKT cell development. We detected selective PLZF the Roquin target ICOS, which, in turn, amplifies expression of downregulation in Roquin-1/2–deficient NKT17 cells and normal c-Maf (56). c-Maf induces an IL-17–producing cell fate, together with levels in the other subsets. This argues against a causal role for its binding partner Sox5t, by directly enhancing RORgt expression 10 ROQUIN PARALOGS POLARIZE NKT CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Analysis of molecules associated with TCR signaling, NKT17 cell differentiation, and apoptosis in Roquin-1/2–deficient NKT cells. (A)Barsshow mean normalized TCRb MFI of the indicated thymic NKT cell populations of control or CD4Cre Rc3h1-2F/F mice. MFI was normalized to TCRb MFIofNKT1 cells of control mice, which was set to 1. Each symbol represents one mouse and shown data were pooled from at least three independent experiments. *p , 0.05, ns, not significant, multiple t tests using the Holm–Sidak method. (B) Representative contour plots show Ly-6C expression on NKT1, NKT2, and NKT17 cells of the indicated genotypes. Shown data were pooled from two independent experiments, and numbers indicate the mean percentage 6 SD of Ly-6C+ cells of at least four mice per genotype. (C) Thymic NKT cells of control and CD4Cre Rc3h1-2F/F mice were analyzed for expression of the intracellular transcription factors Egr2, PLZF, and c-Maf, as well as for phosphorylation of ZAP70, PLCg, and STAT3. Bars indicate mean MFI of the indicated cell population. MFI was normalized to that of TCRb+ tetramer2 cells, which was set to 1. Each data point represents one mouse pooled from at least three independent experiments (PLZF and p-STAT3) or one experiment (Egr2, c-Maf, p-ZAP70, and p-PLCg). Cells were acquired after MACS depletion of CD8+ thymocytes. (D) Bars show mean MFI of intracellular ThPOK expressed in the indicated thymic cell populations isolated from CD4Cre Rc3h1-2F/F mice and controls. Data were pooled from three independent ex- periments, with each data point representing one analyzed mouse. (E) Thymic NKT cells were isolated from control or CD4Cre Rc3h1-2F/F mice and analyzed for cell death by 7-AAD and Annexin V staining. Bars indicate the mean percentage of 7-AAD+ Annexin V+ DP cells. Each data point represents one mouse pooled from three independent experiments. (F) Roquin-deficient NKT cells were analyzed for expression of IL-7R a-chain (CD127). Bars indicate mean MFI 6 SD normalized to TCRb+ tetramer2 cells, which was set to 1. Each data point represents one mouse pooled from two independent experiments. Before acquisition, thymi in (A–C)and(E) were depleted of CD8 by MACS. *p , 0.05, multiple t tests using the Holm–Sidak method. ns, not significant. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 8. Signaling through mTOR does not contribute to the decreased production of subset-specific cytokines of Roquin-1/2–deficient NKT cells. by guest on September 29, 2021 (A) Thymic NKT cells were analyzed for the presence of intracellular p-mTOR, p-AKT (S473), and p-4EBP1 proteins by flow cytometry. Cells were acquired after MACS depletion of CD8+ thymocytes. Bars indicate mean normalized MFIs pooled from two independent experiments (p-mTOR) or from one experiment (p-AKT, p-4EBP1), with each data point representing one analyzed mouse. Multiple t tests using the Holm–Sidak method. (B) Repre- sentative contour plots show cytokine production of thymic NKT1, NKT2, and NKT17 cells isolated from mice of the indicated genotypes. Thymocytes depleted of CD8-expressing cells by MACS were stimulated for 4 h with PMA and ionomycin in the presence of monensin in complete RPMI 1640 medium with 10% FCS. Bar graphs show mean percentages of IFN-g+ NKT1, IL-4+ NKT2, and IL-17+ NKT17 cells, with each data point representing one mouse pooled from a total of three independent experiments. ***p , 0.001, ****p , 0.0001, unpaired t test. (C) Data show mean IL-17 MFI of IL-17+ thymic NKT17 cells of the indicated genotypes after restimulation with PMA and ionomycin for 4 h in the presence of monensin. Each data point represents one mouse; data were pooled from three independent experiments. **p , 0.005, Student t test. (D) Representative contour plots show IFN-g and IL-17 production of CD4+ Foxp32 RORgt+ colon lamina propria lymphocytes of the indicated genotypes. Numbers indicate mean 6 SD calculated from three mice of one experiment. ns, not significant. and by binding to additional gene loci implicated in Th17 differ- Roquin-1/2, all NKT cell subsets showed a decreased production entiation (43), including the Roquin targets Irf4 and Nfkbiz (25). of subset-specific cytokines, which, in the case of NKT17, cor- Further transcriptional activation of these and other target genes relates with reduced STAT3 phosphorylation. NKT cells acquire during initial NKT17 differentiation could then be potentiated by features of memory T cells and innate-like cytokine-production the absence of Roquin-1/2–mediated posttranscriptional control, capabilities during their development, and it appears that they resulting in the production of NKT17 cells at the expense of other become desensitized toward autoantigen recognition during their subsets. development (27). We do not know whether loss of Roquin pro- We cannot provide a conclusive explanation for the absence of teins dampens cytokine production by NKT cells through direct or Roquin-1/2–deficient peripheral NKT cells. The general inflam- indirect means. Upregulation of c-Maf is a consistent feature of matory environment caused by the loss of Roquin paralogs in Roquin-1/2–deficient thymic NKT cells. In addition to promoting T cells could contribute to the loss of Roquin-1/2–deficient NKT cells the differentiation of IL-17–producing T cell subsets, c-Maf is highly in the periphery. The expression of a Va14i TCR and the presence of expressed in exhausted CD4 and CD8 T cells. Overexpression of a majority of wild-type T cells in our mixed bone marrow chimeras c-Maf was sufficient to inhibit CD8 T cell cytokine production and substantially reduce general inflammation. This might partially ex- antitumor responses (57). Therefore, high c-Maf levels could con- plain why mature Roquin-1/2–deficient NKT cells can be maintained tribute to the blunted cytokine responses of Roquin-1/2–deficient in the periphery in both models. NKT cells. Loss of Roquin functionality in conventional T cells leads to NKT cells are a small TCR repertoire–restricted glycolipid- disease-causing effector T cell polarization and excessive cytokine recognizing subset implicated in a large spectrum of human dis- production (20, 22, 25, 38). In striking contrast, after ablation of eases, including infection, autoimmunity, and malignancy. During 12 ROQUIN PARALOGS POLARIZE NKT CELLS their development, they mature into functionally different subsets. 23. Pratama, A., R. R. Ramiscal, D. G. Silva, S. K. Das, V. Athanasopoulos, J. Fitch, N. K. Botelho, P.-P. Chang, X. Hu, J. J. Hogan, et al. 2013. Roquin-2 shares In this article, we show that the posttranscriptional regulators functions with its paralog Roquin-1 in the repression of mRNAs controlling T Roquin-1 and -2 play key cell-intrinsic roles in restricting the follicular helper cells and systemic inflammation. Immunity 38: 669–680. excessive developmental generation of NKT17 cells. 24. Bertossi, A., M. Aichinger, P. Sansonetti, M. Lech, F. Neff, M. Pal, F. T. Wunderlich, H. J. Anders, L. Klein, and M. Schmidt-Supprian. 2011. Loss of Roquin induces early death and immune deregulation but not autoimmunity. J. Acknowledgments Exp. Med. 208: 1749–1756. 25. Jeltsch, K. M., D. Hu, S. Brenner, J. Zo¨ller, G. A. Heinz, D. Nagel, K. U. Vogel, We thank Julia Knogler and Martina Schmickl for technical assistance. N. Rehage, S. C. Warth, S. L. Edelmann, et al. 2014. Cleavage of roquin and Fluorophore-labeled mCD1d PBS-57 tetramers were provided by the Na- regnase-1 by the paracaspase MALT1 releases their cooperatively repressed tional Institutes of Health Tetramer Core Facility. targets to promote T(H)17 differentiation. Nat. Immunol. 15: 1079–1089. 26. Lee, P. P., D. R. Fitzpatrick, C. Beard, H. K. Jessup, S. Lehar, K. W. Makar, M. Pe´rez-Melgosa, M. T. Sweetser, M. S. Schlissel, S. Nguyen, et al. 2001. A Disclosures critical role for Dnmt1 and DNA methylation in T cell development, function, The authors have no financial conflicts of interest. and survival. Immunity 15: 763–774. 27. Vahl, J. C., K. Heger, N. Knies, M. Y. Hein, L. Boon, H. Yagita, B. Polic, and M. Schmidt-Supprian. 2013. NKT cell-TCR expression activates conventional T cells in vivo, but is largely dispensable for mature NKT cell biology. PLoS References Biol. 11: e1001589. 1. Berzins, S. P., M. J. Smyth, and A. G. Baxter. 2011. Presumed guilty: natural 28. Wunderlich, F. T., P. Stro¨hle, A. C. Ko¨nner, S. Gruber, S. Tovar, H. S. Bro¨nneke, killer T cell defects and human disease. Nat. Rev. Immunol. 11: 131–142. L. Juntti-Berggren, L.-S. Li, N. van Rooijen, C. Libert, et al. 2010. Interleukin-6 2. Juno, J. A., Y. Keynan, and K. R. Fowke. 2012. Invariant NKT cells: regulation signaling in liver-parenchymal cells suppresses hepatic inflammation and im- and function during viral infection. PLoS Pathog. 8: e1002838. proves systemic insulin action. Cell Metab. 12: 237–249. 3. Haeryfar, S. M. M., and T. Mallevaey. 2015. Editorial: CD1- and MR1-restricted 29. Westendorf, K., P. Durek, S. Ayew, M.-F. Mashreghi, and A. Radbruch. 2016. Downloaded from T cells in antimicrobial immunity. Front. Immunol. 6: 611. Chromosomal localisation of the CD4cre transgene in B6·Cg-Tg(Cd4-cre)1Cwi 4. Salio, M., J. D. Silk, E. Y. Jones, and V. Cerundolo. 2014. Biology of CD1- and mice. J. Immunol. Methods 436: 54–57. MR1-restricted T cells. Annu. Rev. Immunol. 32: 323–366. 30. Schmidt-Supprian, M., J. Tian, E. P. Grant, M. Pasparakis, R. Maehr, H. Ovaa, 5. Vivier, E., S. Ugolini, D. Blaise, C. Chabannon, and L. Brossay. 2012. Targeting H. L. Ploegh, A. J. Coyle, and K. Rajewsky. 2004. Differential dependence of natural killer cells and natural killer T cells in cancer. Nat. Rev. Immunol. 12: CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF- 239–252. kappaB activation. Proc. Natl. Acad. Sci. USA 101: 4566–4571. 6. Mori, L., M. Lepore, and G. De Libero. 2016. The immunology of CD1- and 31. Egawa, T., G. Eberl, I. Taniuchi, K. Benlagha, F. Geissmann, L. Hennighausen, MR1-restricted T cells. Annu. Rev. Immunol. 34: 479–510. A. Bendelac, and D. R. Littman. 2005. Genetic evidence supporting selection of

7. Heller, F., I. J. Fuss, E. E. Nieuwenhuis, R. S. Blumberg, and W. Strober. 2002. the Valpha14i NKT cell lineage from double-positive thymocyte precursors. http://www.jimmunol.org/ Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated Immunity 22: 705–716. by IL-13–producing NK-T cells. Immunity 17: 629–638. 32. Sun, Z., D. Unutmaz, Y. R. Zou, M. J. Sunshine, A. Pierani, S. Brenner-Morton, 8. Wingender, G., P. Rogers, G. Batzer, M. S. Lee, D. Bai, B. Pei, A. Khurana, R. E. Mebius, and D. R. Littman. 2000. Requirement for RORgamma in thy- M. Kronenberg, and A. A. Horner. 2011. Invariant NKT cells are required for mocyte survival and lymphoid organ development. Science 288: 2369–2373. airway inflammation induced by environmental antigens. J. Exp. Med. 208: 33. Lee, Y. J., K. L. Holzapfel, J. Zhu, S. C. Jameson, and K. A. Hogquist. 2013. 1151–1162. Steady-state production of IL-4 modulates immunity in mouse strains and is 9. Wolf, M. J., A. Adili, K. Piotrowitz, Z. Abdullah, Y. Boege, K. Stemmer, determined by lineage diversity of iNKT cells. Nat. Immunol. 14: 1146–1154. M. Ringelhan, N. Simonavicius, M. Egger, D. Wohlleber, et al. 2014. Metabolic 34. Engel, I., M. Zhao, D. Kappes, I. Taniuchi, and M. Kronenberg. 2012. The activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic transcription factor Th-POK negatively regulates Th17 differentiation in Va14i steatohepatitis and liver cancer via cross-talk with hepatocytes. Cancer Cell 26: NKT cells. Blood 120: 4524–4532. 549–564. 35. Dai, H., A. Rahman, A. Saxena, A. Jaiswal, A. Mohamood, L. Ramirez, S. Noel, 10. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells. R. Hamid, C. Jie, and A. R. Hamad. 2015. Syndecan-1 identifies and controls the by guest on September 29, 2021 Annu. Rev. Immunol. 25: 297–336. frequency of IL-17-producing naı¨ve natural killer T (NKT17) cells in mice. Eur. 11. Matsuda, J. L., T. Mallevaey, J. Scott-Browne, and L. Gapin. 2008. CD1d- J. Immunol. 45: 3045–3051. restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system. Curr. 36. Milpied, P., B. Massot, A. Renand, S. Diem, A. Herbelin, M. Leite-de-Moraes, Opin. Immunol. 20: 358–368. M. T. Rubio, and O. Hermine. 2011. IL-17-producing invariant NKT cells in 12. Savage, A. K., M. G. Constantinides, J. Han, D. Picard, E. Martin, B. Li, lymphoid organs are recent thymic emigrants identified by neuropilin-1 ex- O. Lantz, and A. Bendelac. 2008. The transcription factor PLZF directs the pression. Blood 118: 2993–3002. effector program of the NKT cell lineage. Immunity 29: 391–403. 37. Griewank, K., C. Borowski, S. Rietdijk, N. Wang, A. Julien, D. G. Wei, 13. Kovalovsky, D., U. O. Uche, S. Eladad, R. M. Hobba, W. Yi, E. Alonzo, A. A. Mamchak, C. Terhorst, and A. Bendelac. 2007. Homotypic interactions K. Chua, M. Eidson, H.-J. Kim, J. S. Im, and P. P. Pandolfi. 2008. The BTB–zinc mediated by Slamf1 and Slamf6 receptors control NKT cell lineage develop- finger transcriptional regulator PLZF controls the development of invariant ment. Immunity 27: 751–762. natural killer T cell effector functions. Nat. Immunol. 9: 1055–1064. 38. Vinuesa, C. G., M. C. Cook, C. Angelucci, V. Athanasopoulos, L. Rui, 14. Raberger, J., A. Schebesta, S. Sakaguchi, N. Boucheron, K. E. M. Blomberg, K. M. Hill, D. Yu, H. Domaschenz, B. Whittle, T. Lambe, et al. 2005. A RING- A. Berglo¨f, T. Kolbe, C. I. E. Smith, T. Rulicke,€ and W. Ellmeier. 2008. The type ubiquitin ligase family member required to repress follicular helper T cells transcriptional regulator PLZF induces the development of CD44 high memory and autoimmunity. Nature 435: 452–458. phenotype T cells. Proc. Natl. Acad. Sci. USA 105: 17919–17924. 39. Martin, B., C. Auffray, A. Delpoux, A. Pommier, A. Durand, C. Charvet, 15. Kovalovsky, D., E. S. Alonzo, O. U. Uche, M. Eidson, K. E. Nichols, and P. Yakonowsky, H. de Boysson, N. Bonilla, A. Audemard, et al. 2013. Highly D. B. Sant’Angelo. 2010. PLZF induces the spontaneous acquisition of memory/ self-reactive naive CD4 T cells are prone to differentiate into regulatory T cells. effector functions in T cells independently of NKT cell-related signals. J. Immunol. Nat. Commun. 4: 2209. 184: 6746–6755. 40. Lazarevic, V., A. J. Zullo, M. N. Schweitzer, T. L. Staton, E. M. Gallo, 16. Godfrey, D. I., S. Stankovic, and A. G. Baxter. 2010. Raising the NKT cell G. R. Crabtree, and L. H. Glimcher. 2009. The gene encoding early growth family. Nat. Immunol. 11: 197–206. response 2, a target of the transcription factor NFAT, is required for the devel- 17. Benlagha, K., D. G. Wei, J. Veiga, L. Teyton, and A. Bendelac. 2005. Characterization opment and maturation of natural killer T cells. Nat. Immunol. 10: 306–313. of the early stages of thymic NKT cell development. J. Exp. Med. 202: 485–492. 41. Gleimer, M., H. von Boehmer, and T. Kreslavsky. 2012. PLZF controls the 18. Gapin, L. 2016. Development of invariant natural killer T cells. Curr. Opin. expression of a limited number of genes essential for NKT cell function. Front. Immunol. 39: 68–74. Immunol. 3: 374. 19. Engel, I., G. Seumois, L. Chavez, D. Samaniego-Castruita, B. White, A. Chawla, 42. Bauquet, A. T., H. Jin, A. M. Paterson, M. Mitsdoerffer, I.-C. Ho, A. H. Sharpe, D. Mock, P. Vijayanand, and M. Kronenberg. 2016. Innate-like functions of and V. K. Kuchroo. 2009. The costimulatory molecule ICOS regulates the ex- natural killer T cell subsets result from highly divergent gene programs. Nat. pression of c-Maf and IL-21 in the development of follicular T helper cells and Immunol. 17: 728–739. TH-17 cells. Nat. Immunol. 10: 167–175. 20. Yu, D., A. H. Tan, X. Hu, V. Athanasopoulos, N. Simpson, D. G. Silva, 43. Tanaka, S., A. Suto, T. Iwamoto, D. Kashiwakuma, S. Kagami, K. Suzuki, A. Hutloff, K. M. Giles, P. J. Leedman, K. P. Lam, et al. 2007. Roquin represses H. Takatori, T. Tamachi, K. Hirose, A. Onodera, et al. 2014. Sox5 and c-Maf autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. cooperatively induce Th17 cell differentiation via RORgt induction as down- [Published erratum appears in 2008 Nature 451: 1022.] Nature 450: 299–303. stream targets of Stat3. J. Exp. Med. 211: 1857–1874. 21. Glasmacher, E., K. P. Hoefig, K. U. Vogel, N. Rath, L. Du, C. Wolf, E. Kremmer, 44. Yang, Y., J. Ochando, A. Yopp, J. S. Bromberg, and Y. Ding. 2005. IL-6 plays a X. Wang, and V. Heissmeyer. 2010. Roquin binds inducible costimulator mRNA unique role in initiating c-Maf expression during early stage of CD4 T cell and effectors of mRNA decay to induce microRNA-independent post- activation. J. Immunol. 174: 2720–2729. transcriptional repression. Nat. Immunol. 11: 725–733. 45.Rachitskaya,A.V.,A.M.Hansen,R.Horai,Z.Li,R.Villasmil,D.Luger, 22. Vogel, K. U., S. L. Edelmann, K. M. Jeltsch, A. Bertossi, K. Heger, G. A. Heinz, R. B. Nussenblatt, and R. R. Caspi. 2008. Cutting edge: NKT cells constitu- J. Zo¨ller, S. C. Warth, K. P. Hoefig, C. Lohs, et al. 2013. Roquin paralogs 1 and 2 tively express IL-23 receptor and RORgammat and rapidly produce IL-17 redundantly repress the Icos and Ox40 costimulator mRNAs and control fol- upon receptor ligation in an IL-6-independent fashion. J. Immunol. 180: licular helper T cell differentiation. Immunity 38: 655–668. 5167–5171. The Journal of Immunology 13

46. Enders, A., S. Stankovic, C. Teh, A. P. Uldrich, M. Yabas, T. Juelich, J. A. Altin, and A. Singer. 2015. Let-7 microRNAs target the lineage-specific transcription S. Frankenreiter, H. Bergmann, C. M. Roots, et al. 2012. ZBTB7B (Th-POK) factor PLZF to regulate terminal NKT cell differentiation and effector function. regulates the development of IL-17–producing CD1d-restricted mouse Nat. Immunol. 16: 517–524. NKT cells. J. Immunol. 189: 5240–5249. 53. Engel, I., K. Hammond, B. A. Sullivan, X. He, I. Taniuchi, D. Kappes, and 47. Tani-ichi, S., A. Shimba, K. Wagatsuma, H. Miyachi, S. Kitano, K. Imai, M. Kronenberg. 2010. Co-receptor choice by V a14i NKT cells is driven by Th- T. Hara, and K. Ikuta. 2013. Interleukin-7 receptor controls development and POK expression rather than avoidance of CD8-mediated negative selection. maturation of late stages of thymocyte subpopulations. Proc. Natl. Acad. Sci. J. Exp. Med. 207: 1015–1029. USA 110: 612–617. 54. Wang, L., T. Carr, Y. Xiong, K. F. Wildt, J. Zhu, L. Feigenbaum, A. Bendelac, 48. Webster, K. E., H.-O. Kim, K. Kyparissoudis, T. M. Corpuz, G. V. Pinget, and R. Bosselut. 2010. The sequential activity of Gata3 and Thpok is required for A. P. Uldrich, R. Brink, G. T. Belz, J. H. Cho, D. I. Godfrey, and J. Sprent. 2014. the differentiation of CD1d-restricted CD4+ NKT cells. Eur. J. Immunol. 40: IL-17-producing NKT cells depend exclusively on IL-7 for homeostasis and 2385–2390. survival. Mucosal Immunol. 7: 1058–1067. 55. Rutz, S., R. Noubade, C. Eidenschenk, N. Ota, W. Zeng, Y. Zheng, J. Hackney, 49. Yang, W., B. Gorentla, X.-P. Zhong, and J. Shin. 2015. mTOR and its tight J. Ding, H. Singh, and W. Ouyang. 2011. Transcription factor c-Maf mediates the regulation for iNKT cell development and effector function. Mol. Immunol. 68(2 TGF-b–dependent suppression of IL-22 production in T(H)17 cells. Nat. Pt. C): 536–545. Immunol. 12: 1238–1245. 50. Wu, J., J. Yang, K. Yang, H. Wang, B. Gorentla, J. Shin, Y. Qiu, L. G. Que, 56. Pot, C., H. Jin, A. Awasthi, S. M. Liu, C.-Y. Lai, R. Madan, A. H. Sharpe, W. M. Foster, Z. Xia, et al. 2014. iNKT cells require TSC1 for terminal matu- C. L. Karp, S.-C. Miaw, I.-C. Ho, and V. K. Kuchroo. 2009. Cutting edge: IL-27 ration and effector lineage fate decisions. J. Clin. Invest. 124: 1685–1698. induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory 51. Chang, P.-P., S. K. Lee, X. Hu, G. Davey, G. Duan, J.-H. Cho, G. Karupiah, receptor ICOS that coordinately act together to promote differentiation of IL-10– J. Sprent, W. R. Heath, E. M. Bertram, and C. G. Vinuesa. 2012. Breakdown in producing Tr1 cells. J. Immunol. 183: 797–801. repression of IFN-g mRNA leads to accumulation of self-reactive effector CD8+ 57. Giordano, M., C. Henin, J. Maurizio, C. Imbratta, P. Bourdely, M. Buferne, T cells. J. Immunol. 189: 701–710. L. Baitsch, L. Vanhille, M. H. Sieweke, D. E. Speiser, et al. 2015. Molecular 52. Pobezinsky, L. A., R. Etzensperger, S. Jeurling, A. Alag, T. Kadakia, profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of T. M. McCaughtry, M. Y. Kimura, S. O. Sharrow, T. I. Guinter, L. Feigenbaum, exhaustion. EMBO J. 34: 2042–2058. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021