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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Curr Opin Immunol. 2013 Feb 9;25(2):161–167. doi: 10.1016/j.coi.2013.01.003

Trancriptional regulation of the NKT cell lineage

Michael G Constantinides 1, Albert Bendelac 1
PMCID: PMC3647452  NIHMSID: NIHMS444429  PMID: 23402834

Abstract

How expression of canonical semi-invariant TCRs leads to innate-like effector differentiation is a central enigma of NKT cell development. NKT thymic precursors undergo elevated TCR signals leading to increased Egr2, which directly induces their signature transcription factor, PLZF. PLZF is necessary and sufficient to induce a multipotent, unbiased effector program that precedes terminal differentiation into T-bethigh NK1.1+ (NKT1) cells and recently identified NKT2 and NKT17 sublineages. Major variations in polarized NKT sublineages have been uncovered in different mouse strains and in several mutants, with direct impact on NKT cell function but also, unexpectedly, on the development and function of conventional T cells.

Introduction

Innate-like T cell lineages are defined by their evolutionarily conserved, semi-invariant αβ or γδ TCRs and their expression of the signature transcription factor PLZF [1,2]. The best characterized population is composed of αβ T cells specific for microbial lipids presented by MHC class I-like CD1d molecules, which mostly (but not exclusively) express canonical Vα14-Jα18 TCRα chains associated with Vβ8, Vβ7 or Vβ2 [3,4]. Other PLZF-expressing lineages include Vγ1-Vδ6.3 T cells [5,6], whose ligand is unknown, and mucosal associated invariant T (MAIT) cells expressing Vα19-Jα33 TCRs specific for microbial vitamin B metabolites presented by MR1 [7,8].

Because expression of these canonical TCRs is sufficient to induce PLZF expression and innate-like differentiation [5,9], studies of TCR signaling and co-signaling events during thymic selection have provided critical insights into the mechanisms of NKT lineage decision. Recent advances in this area are the subject of this review.

Elevated TCR signaling during thymic selection

The Vα14 TCR recognizes several endogenous agonist lipid ligands [10–13] presented by CD1d on the surface of cortical thymocytes [14]. Accordingly, various studies support the notion that NKT thymocytes undergo elevated TCR signaling. For example, although NKT precursors activate Ras/MAP kinase [15] and calcineurin/NFAT [16] as conventional thymocytes, they exhibit more elevated Egr1 and Egr2 downstream of these respective pathways at their CD69+CD24high ‘transitional’ stage following TCR engagement (so-called ‘stage 0’) compared with conventional thymocytes, and these levels are sustained in later stages [17]. Consistent with increased TCR signaling, stage 0 and stage 1 cells also express higher levels of CD5 and PD-1 and they express more GFP in Nur77-GFP reporter mice [18].

How NKT precursors escape negative selection despite agonist signaling may be due to the lack of B7 expression by cortical thymocytes [19,20] and to the alternative engagement of homophilic interactions between Slam family molecules such as Slamf1 and Slamf6 [21], which signal through SAP and Fyn to induce NF-kB and promote NKT cell survival.

PLZF expression induced by Egr2

PLZF induction begins at stage 0, when Egr2 levels are highest, and is expressed by 100% of NKT thymocytes at the subsequent stage. Egr2 ChIP-Seq of NKT thymocytes revealed binding of Egr2 to a single canonical Egr binding site in the proximal promoter of PLZF [17]. Furthermore, injection of anti-TCRβ antibody as an agonist ligand in vivo led to an immediate increase of Egr2 followed by PLZF mRNA induction in conventional thymocytes. Other Egrs may also bind the PLZF promoter and it is difficult to dissect their respective contribution because Egr2-deficient thymocytes had a compensatory increase in Egr1. Nevertheless, Egr1/2 double deficient NKT thymocytes did not express PLZF at stage 1 and, likewise, injection of anti-TCRβ antibody in these double mutant mice failed to induce PLZF in thymocytes [17]. The induction of PLZF as a consequence of elevated TCR signaling and Egr2 may be a general feature of innate-like T cell lineages, as Vγ expressing, anti-TCR antibodies induced PLZF expression during development of the corresponding Vγ-expressing ubsetin OP9-DL1 cultures [5]. Furthermore, in a model of transgenic MHC class II expression on cortical thymocytes where MHC class II-restricted CD4 T cells acquire innate-like effector differentiation, PLZF expression was observed on the fraction of cells with the highest level of Nur77-GFP [22].

Egr2 and PLZF direct different branches of NKT lineage differentiation

In general, Egr2 appears to promote cell division and survival through direct activation of Ccnd2, Bcl2 and Fasl [17], while PLZF directs the acquisition of effector properties [23]. In the absence of PLZF, NKT cells revert to naïve-like cells recirculating between lymph nodes and blood and producing IL-2 rather than IL-4 or IFN-γ. Conversely, transgenic expression of PLZF under the Cd4 promoter is sufficient to induce the upregulation of CD44 and the downregulation of CD62L, the upregulation of LFA-1 and the homing to liver and lung, the ability to secrete a mixture of IL-4 and IFN-γ at the single cell level as well as IL-17 [1,2,23–25]. Thus, PLZF induces a mixed, unpolarized cytokine program. PLZF is a member of the BTB-ZF family of transcription factors that binds a corepressor complex containing NCor, Sin3a and HDAC1, as well as the E3 ligase cullin3 whose ablation phenocopies the PLZF deficiency [26]. The molecular details of the transcriptional program and the direct targets of PLZF are under investigation. Nevertheless, it is notable that this program can unfold in the absence of cell division or agonist signaling, as shown in PLZF transgenic mice, demonstrating that acquisition of effector properties can be uncoupled from strong TCR signaling and division [23,24]. Consistent with this view, PLZF-deficient NKT thymocytes still exhibit the same rate of EdU incorporation as their wild-type counterparts [1,2], which is presumably driven by Egr2 and other TCR signals. However, the mutant cells do not expand, suggesting increased cell death. In that regard, while anti-apoptotic signals arise from Slamf-SAP-Fyn-PKCΘ-NF-κB signaling, PLZF may also exert anti-apoptotic properties, as suggested by the partial rescue of Mtv-reactive thymocytes from negative selection in PLZF transgenic mice [23]. Importantly, Egr2 appears to contribute to the initiation of NKT1 polarization as it plays an essential role in binding and activating Il2rb [17], a key component of the IL-15 receptor that is critical to this terminal differentiation pathway. Consistent with this role of Egr2, low levels of IL-2Rβ and NK1.1 can be observed in a fraction of PLZF-deficient NKT thymocytes [1,2]. Furthermore, it has been reported that continuous exposure to CD1d was required for NK1.1 expression [27,28], which may be due to the need for sustained Egr2.

Terminal differentiation of polarized NKT sublineages

T-bethigh NKT1 cells capable of producing large amounts of IFN-γ are the main terminal differentiation product in B6 mice [27,29]. It should be stressed, however, that these cells are not fully polarized in that they produce both IFN-γ and IL-4 on a single cell basis, although the amount of IL-4 is comparatively low compared with previous developmental stages. Furthermore, it is the NK1.1+ subset that is responsible for the rapid release of IL-4 upon stimulation of NKT cells in vivo [30]. Recent studies, however, have uncovered alternatively polarized NKT sublineages, including GATA3high NKT2 [31,32] and RORγt+ NKT17 cells [33–35]. These sublineages are rare in B6 mice but can be dominant in other strains or in various mutant mice. NKT2 cells, which are more abundant in BALB/c mice, predominate in the lung; NKT17 cells are rare and mostly found in peripheral lymph nodes draining the skin; NKT1 cells predominate in spleen and liver. These polarized sublineages have the typical profiles of transcription factors, cytokines and chemokine receptors found in their Th1/Th2/Th17 or ILC1/ILC2/ILC22 counterparts, although their bias seems to be less absolute because, for example, NKT1 cells also produce IL-4.

These new findings warrant a modification of the early model of NKT cell development and raise the question of the branch point at which NKT2 and NKT17 cells emerge. Intracellular staining for GATA3 and RORγt indicate that these subsets are included within the previously termed stage 2 NKT thymocytes, which also contain the precursors to NKT1 cells, as demonstrated by intrathymic cell transfers. In search of a surface marker differentiating true stage 2 precursors from terminally differentiated NKT2 and NKT17, it has been suggested that IL-17RB, a key component of the receptor for IL-25, was selectively expressed in NKT2 and NKT17 precursors but not in NKT1 precursors [32]. These conclusions were derived based on transfers of NKT thymocyte subsets into D-guanosine treated fetal thymus cultured in vitro, or intravenously into Jα18-deficient mice. Strikingly, mice lacking IL-17RB lacked both NKT2 and NKT17, but not NKT1 thymocytes, suggesting that commitment to these sublineages required signaling through this receptor [32](Dr. Watarai, personal communication). As IL-17RB is expressed as early at in stage 1 cells, the commitment to NKT1 vs NKT2 or 17 sublineages may occur very early. Because ~50% of stage 1 and 75% of stage 2 cells are IL-17RB+ in the B6 background, the implication of these findings is that there are more thymic precursors to the NKT2 and NKT17 lineage than to the NKT1 lineage, which is in apparent conflict with the overall predominance of mature NKT1 cells in B6 mice both in thymus and in periphery. One possibility is that the NKT2 and NKT17 cells are short-lived compared with NKT1 cells, which exhibit IL-15-driven self-renewing properties [36]. Indeed, most NKT17 cells in the periphery express neuropilin 1, a marker of recent thymic NKT emigrants, and rapidly disappear after thymectomy [37].

Transcriptional regulation of polarized NKT sublineages

Studies of mice lacking T-bet, GATA3 and RORγt, the signature transcription factors of NKT sublineages have shed partial light on the mechanisms regulating the NKT sublineage decisions. T-bet-deficient [38] and IL-15-deficient mice [36] exhibited a block in the development of NKT1 thymocytes at a IL-2Rβ-NK1.1− and IL-2RβlowNK1.1low stage 2, respectively. Interestingly, they also exhibited a relative increase in NKT2/NKT17 cells [32,38]. In mice lacking the BLIMP-1 homolog Hobit, which is specifically expressed in mouse NKT1 cells, a relative accumulation of NKT2 or stage 2 thymocytes was observed, along with defects in NKT1 cell number and function affecting expression of Ly49 receptors, granzyme B and IFN-γ [39]. Whether the apparent crossregulation between NKT subsets reflects accumulation of arrested precursors or antagonism between the different sublineages remains to be investigated.

The respective impact of GATA3 and RORγt has been more difficult to evaluate because these mutations have widespread effects on thymocyte development. For example, in the absence of RORγt, distal Vα14-Jα18 rearrangements did not occur, due to the shorter lifespan of cortical thymocytes, and NKT cells were consequently totally absent [40,41]. GATA3fl/fl Cd4-cre mice exhibited a complex phenotype whereby stage 1 and 2 thymocytes were drastically diminished, despite the presence of apparently normal numbers of NKT1 cells in the thymus [42]. These NKT1 cells were not merely cells that escaped GATA3 deletion and accumulated in the thymus, because their expression of ThPOK, a target of GATA3, was also impaired [43]. There were fewer and dysfunctional NKT1 cells in the periphery, however, implying that GATA3 impacted the development and/or survival of all NKT sublineages.

As transgenic studies have established that PLZF expression is sufficient to prime developing thymocytes for the production of an unbiased mixture of IL-4, IFN-γ and IL-17, an appealing scenario is that PLZF promotes a mixed pattern of gene expression by multilineage transcriptional priming, similar to PU-1 in myeloid progenitors [44], prior to the segregation of polarized transcriptional programs driven by antagonistic regulatory circuits. In that regard, it is intriguing that PLZF is partially downregulated in NKT17 (PLZFint) and NKT1 (PLZFlow) compared with early NKT precursors and NKT2 cells (PLZFhigh). As previous reports have ruled out a role for STAT4 and STAT6 in NKT cell development [45,46], novel mechanisms that determine the terminal polarization of NKT1, NKT2 and NKT17 remain to be elucidated.

Dysregulation of NKT2 and NKT17 sublineages in inbred and mutant strains

Intriguingly, several apparently unrelated mutations were recently found to produce closely related phenotypes consisting of the transformation of conventional naïve thymocytes into effector/memory cells prior to emigration. Hogquist et al. investigated the phenotype of mice lacking KLF-2, a transcription factor that controls CD62L and S1P1 expression after positive selection and was previously associated with the maintenance of lymphocyte quiescence. Mixed bone marrow chimeras established that the ‘innate effector/memory’ phenotype of conventional T cells in KLF2 deficient mice was in fact the bystander consequence of a cell-intrinsic dysregulation of NKT2 (or stage 2) NKT thymocytes releasing IL-4 in the steady state [47,48]. Furthermore, an expansion of IL-4 producing Vγ1-Vδ6.3 T cells was also observed, indicating a general dysregulation of PLZF-expressing thymic lineages [49]. A similar mechanism operates in mice lacking the Tec kinases Itk or Rlk [47,50,51], or bearing the Y146F mutation in the scaffold protein SLP756 [6], which impairs the binding of these kinases after TCR signaling, as well as in mice lacking the E protein inhibitor Id3 [6,52,53] or the histone acetyl transferase CBP [54]. At present, it is unclear whether these genes belong to the same signaling pathway or whether they impact different regulatory pathways that ultimately converge to cause an expansion of NKT2-like cells and the steady-state secretion of IL-4.

While the physiological significance of these mutations is unclear, it is remarkable that a related phenotype, including the effector/memory conversion of thymic CD8 cells, was recently reported in wild-type BALB/c mice, which harbor a spontaneous expansion of NKT2 cells [47,55]. In the absence of CD1d, BALB/c CD8 T cells reverted to a naïve phenotype, demonstrating that NKT cells exert bystander regulation of the thymic microenvironment with substantial impact on T cell functional properties. Notably, BALB/c NKT thymocytes lost their exaggerated NKT2 phenotype in the absence of the transcription factor KLF-13, although this factor did not appear to be dysregulated in BALB/c cells [55].

ThPOK, a critical regulator of the CD4 lineage is also expressed in all NKT cells, whether CD4+ or CD4−, but is not required for their development. However, closer analysis revealed that ThPOK-deficient mice exhibited not only partial defects in NKT1 differentiation and frequency, but also a reciprocal increase in RORγt-expressing NKT17-like cells, suggesting a degree of antagonism between ThPOK and RORγt [43,56,57].

NKT-fh and NKT-reg

NKT cells can readily express Bcl-6 and become follicular helper cells upon activation by their microbial lipid ligands [58,59], and they can express Foxp3 and suppress CD4 T cell proliferation following treatment with TGFβ [60,61]. However, these pathways do not seem to operate during development in the thymus, at least in normal mice.

Role of the PI3K/mTOR pathway

As NKT thymocytes, unlike conventional CD4 and CD8 T cells, undergo blasting and cell division during development, it is anticipated that their metabolic needs will be tightly regulated by the PI3K/mTOR pathway. Mice lacking, or expressing a catalytically inactive form of phosphoinositide dependent kinase 1 (PDK1), which is required for activation of Akt showed NKT developmental arrest between stages 0 and 1, with defective blasting and curtailed expansion of NKT precursors [62], a phenotype similar to the c-Myc mutant NKT thymocytes [63,64]. Mice lacking Tsc-1, a suppressor of mTORC1, exhibited defective NKT cell development due to apoptosis [65]. How the mTOR pathway is recruited and whether it can also differentially regulate the polarized NKT sublineages, as is the case for T-helper lineages [66], are areas of investigation.

Conclusion

Recent advances have elucidated, in great detail, the TCR signaling pathway and the transcriptional network governing NKT lineage development. The classic ‘stage 0-1-2-3’ model has been enriched by the discovery of alternative NKT2 and NKT17 sublineages which not only impact NKT cell responses to microbial pathogens, but also regulate in the steady state previously unsuspected aspects of conventional T cell development and function. Many new exciting areas are open for future investigations, including a genomic and biochemical understanding of PLZF function with identification of its direct targets and its broader transcriptional regulatory network. Multilineage priming by a BTB-ZF transcription factor and its subsequent resolution into cytokine polarized sublineages, as apparent in PLZF-expressing innate-like T cell lineages, may be a shared mechanism across lymphoid cells such as CD4 T helper cells and innate lymphoid cells.

Figure 1. Thymic developmental pathway of the NKT cell lineage.

Figure 1

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The current model incorporates the newly discovered NKT2 and NKT17 subsets side by side with NKT1 cells (all of which correspond to developmental stage 3). The branching points between the NKT1, NKT2 and NKT17 sublineages at stage 1 and 2 are still under study. The thymic emigrants are still cycling and immature. Note, that Tbet+ NKT1 cells do produce IL4, albeit in smaller amounts than NKT2 cells. Some NKT1 precursors fail to emigrate, possibly due to premature downregulation of S1P1 [67], and they accumulate in the thymus of aging mice (not depicted). NKR, NK lineage receptors including NK1.1, Ly49, NKG2D, CD94, DX5.

Highlights.

  • PLZF is the master transcription factor of the NKT cell lineage.

  • PLZF is induced during thymic development by Egr2 downstream of TCR/NFAT signaling.

  • PLZF directs acquisition of the innate-like effector program whereas Egr2 and other TCR signals/co-signals induce cell division.

  • In addition to the major T-bethigh NKT1 subset, novel NKT sublineages include NKT2 and NKT17.

  • Different mouse strains exhibit wide variations in the relative representation of NKT1, NKT2 and NKT17.

Acknowledgments

The authors gratefully acknowledge current and past members of their laboratory for discussions and experiments. Research in the laboratory was supported by NIH PO1 AI053725 and AI038339 to AB and the University of Chicago Digestive Diseases Research Core Center (P30 DK42086). AB is an investigator of the Howard Hughes Medical Institute.

Footnotes

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References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

* of special interest

** of outstanding interest

  • **1.Savage AK, Constantinides MG, Han J, Picard D, Martin E, Li B, Lantz O, Bendelac A. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity. 2008;29:391–403. doi: 10.1016/j.immuni.2008.07.011. This and the next reference represent the first identification of the signature transcription factor PLZF and the characterization of its role in directing innate-like effector differentiation. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • **2.Kovalovsky D, Uche OU, Eladad S, Hobbs RM, Yi W, Alonzo E, Chua K, Eidson M, Kim HJ, Im JS, et al. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nat Immunol. 2008;9:1055–1064. doi: 10.1038/ni.1641. Same comment as above. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol. 2007;25:297–336. doi: 10.1146/annurev.immunol.25.022106.141711. [DOI] [PubMed] [Google Scholar]
  • 4.Godfrey DI, Stankovic S, Baxter AG. Raising the NKT cell family. Nat Immunol. 2010;11:197–206. doi: 10.1038/ni.1841. [DOI] [PubMed] [Google Scholar]
  • 5.Kreslavsky T, Savage AK, Hobbs R, Gounari F, Bronson R, Pereira P, Pandolfi PP, Bendelac A, von Boehmer H. TCR-inducible PLZF transcription factor required for innate phenotype of a subset of gammadelta T cells with restricted TCR diversity. Proc Natl Acad Sci U S A. 2009;106:12453–12458. doi: 10.1073/pnas.0903895106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Alonzo ES, Gottschalk RA, Das J, Egawa T, Hobbs RM, Pandolfi PP, Pereira P, Nichols KE, Koretzky GA, Jordan MS, et al. Development of promyelocytic zinc finger and ThPOK-expressing innate gamma delta T cells is controlled by strength of TCR signaling and Id3. J Immunol. 2010;184:1268–1279. doi: 10.4049/jimmunol.0903218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Le Bourhis L, Guerri L, Dusseaux M, Martin E, Soudais C, Lantz O. Mucosal-associated invariant T cells: unconventional development and function. Trends Immunol. 2011;32:212–218. doi: 10.1016/j.it.2011.02.005. [DOI] [PubMed] [Google Scholar]
  • 8.Kjer-Nielsen L, Patel O, Corbett AJ, Le Nours J, Meehan B, Liu L, Bhati M, Chen Z, Kostenko L, Reantragoon R, et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature. 2012 doi: 10.1038/nature11605. [DOI] [PubMed] [Google Scholar]
  • 9.Bendelac A, Hunziker RD, Lantz O. Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells. J Exp Med. 1996;184:1285–1293. doi: 10.1084/jem.184.4.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Zhou D, Mattner J, Cantu C, 3rd, Schrantz N, Yin N, Gao Y, Sagiv Y, Hudspeth K, Wu YP, Yamashita T, et al. Lysosomal glycosphingolipid recognition by NKT cells. Science. 2004;306:1786–1789. doi: 10.1126/science.1103440. [DOI] [PubMed] [Google Scholar]
  • 11.Facciotti F, Ramanjaneyulu GS, Lepore M, Sansano S, Cavallari M, Kistowska M, Forss-Petter S, Ni G, Colone A, Singhal A, et al. Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus. Nat Immunol. 2012;13:474–480. doi: 10.1038/ni.2245. [DOI] [PubMed] [Google Scholar]
  • 12.Brennan PJ, Tatituri RV, Brigl M, Kim EY, Tuli A, Sanderson JP, Gadola SD, Hsu FF, Besra GS, Brenner MB. Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals. Nat Immunol. 2011;12:1202–1211. doi: 10.1038/ni.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Parekh VV, Singh AK, Wilson MT, Olivares-Villagomez D, Bezbradica JS, Inazawa H, Ehara H, Sakai T, Serizawa I, Wu L, et al. Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids. J Immunol. 2004;173:3693–3706. doi: 10.4049/jimmunol.173.6.3693. [DOI] [PubMed] [Google Scholar]
  • 14.Bendelac A. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J Exp Med. 1995;182:2091–2096. doi: 10.1084/jem.182.6.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hu T, Gimferrer I, Simmons A, Wiest D, Alberola-Ila J. The Ras/MAPK pathway is required for generation of iNKT cells. PLoS One. 2011;6:e19890. doi: 10.1371/journal.pone.0019890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • **16.Lazarevic V, Zullo AJ, Schweitzer MN, Staton TL, Gallo EM, Crabtree GR, Glimcher LH. The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells. Nat Immunol. 2009;10:306–313. doi: 10.1038/ni.1696. This study identifies the requirement of Egr2 downstream of TCR-NFAT signaling for NKT cell development. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • **17.Seiler MP, Mathew R, Liszewski MK, Spooner C, Barr K, Meng F, Singh H, Bendelac A. Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling. Nat Immunol. 2012;13:264–271. doi: 10.1038/ni.2230. This study demonstrates elevated and sustained TCR/Egr signaling in NKT precursors and characterizes the direct role of Egr2 for induction of the lineage-determining transcription factor PLZF and of IL-2Rβ. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Moran AE, Holzapfel KL, Xing Y, Cunningham NR, Maltzman JS, Punt J, Hogquist KA. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J Exp Med. 2011;208:1279–1289. doi: 10.1084/jem.20110308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Graham DB, Bell MP, Huntoon CJ, Griffin MD, Tai X, Singer A, McKean DJ. CD28 ligation costimulates cell death but not maturation of double-positive thymocytes due to defective ERK MAPK signaling. J Immunol. 2006;177:6098–6107. doi: 10.4049/jimmunol.177.9.6098. [DOI] [PubMed] [Google Scholar]
  • 20.Williams JA, Lumsden JM, Yu X, Feigenbaum L, Zhang J, Steinberg SM, Hodes RJ. Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway. J Immunol. 2008;181:907–917. doi: 10.4049/jimmunol.181.2.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Griewank K, Borowski C, Rietdijk S, Wang N, Julien A, Wei DG, Mamchak AA, Terhorst C, Bendelac A. Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development. Immunity. 2007;27:751–762. doi: 10.1016/j.immuni.2007.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Qiao Y, Zhu L, Sofi H, Lapinski PE, Horai R, Mueller K, Stritesky GL, He X, Teh HS, Wiest DL, et al. Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells. Proc Natl Acad Sci U S A. 2012;109:16264–16269. doi: 10.1073/pnas.1207528109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Savage AK, Constantinides MG, Bendelac A. Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling. J Immunol. 2011;186:5801–5806. doi: 10.4049/jimmunol.1100119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kovalovsky D, Alonzo ES, Uche OU, Eidson M, Nichols KE, Sant’Angelo DB. PLZF induces the spontaneous acquisition of memory/effector functions in T cells independently of NKT cell-related signals. J Immunol. 2010;184:6746–6755. doi: 10.4049/jimmunol.1000776. [DOI] [PubMed] [Google Scholar]
  • 25.Thomas SY, Scanlon ST, Griewank KG, Constantinides MG, Savage AK, Barr KA, Meng F, Luster AD, Bendelac A. PLZF induces an intravascular surveillance program mediated by long-lived LFA-1-ICAM-1 interactions. J Exp Med. 2011;208:1179–1188. doi: 10.1084/jem.20102630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • **26.Mathew R, Seiler MP, Scanlon ST, Mao AP, Constantinides MG, Bertozzi-Villa C, Singer JD, Bendelac A. BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs. Nature. 2012 doi: 10.1038/nature11548. [Epub ahead of print]. Characterization of the PLZF transcriptional complex and associated E3 ligase cullin 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.McNab FW, Berzins SP, Pellicci DG, Kyparissoudis K, Field K, Smyth MJ, Godfrey DI. The influence of CD1d in postselection NKT cell maturation and homeostasis. J Immunol. 2005;175:3762–3768. doi: 10.4049/jimmunol.175.6.3762. [DOI] [PubMed] [Google Scholar]
  • 28.Wei DG, Curran SA, Savage PB, Teyton L, Bendelac A. Mechanisms imposing the Vbeta bias of Valpha14 natural killer T cells and consequences for microbial glycolipid recognition. J Exp Med. 2006;203:1197–1207. doi: 10.1084/jem.20060418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A. A thymic precursor to the NKT cell lineage. Science. 2002;296:553–555. doi: 10.1126/science.1069017. [DOI] [PubMed] [Google Scholar]
  • 30.Yoshimoto T, Paul WE. CD4pos NK1.1pos T cells promptly produced IL-4 in response to in vivo challenge with anti-CD3. J Exp Med. 1994;179:1285. doi: 10.1084/jem.179.4.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Terashima A, Watarai H, Inoue S, Sekine E, Nakagawa R, Hase K, Iwamura C, Nakajima H, Nakayama T, Taniguchi M. A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity. J Exp Med. 2008;205:2727–2733. doi: 10.1084/jem.20080698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • **32.Watarai H, Sekine-Kondo E, Shigeura T, Motomura Y, Yasuda T, Satoh R, Yoshida H, Kubo M, Kawamoto H, Koseki H, et al. Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines. PLoS Biol. 2012;10:e1001255. doi: 10.1371/journal.pbio.1001255. Identification of the new NKT2 sublineage. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Doisne JM, Becourt C, Amniai L, Duarte N, Le Luduec JB, Eberl G, Benlagha K. Skin and peripheral lymph node invariant NKT cells are mainly retinoic acid receptor-related orphan receptor (gamma)t+ and respond preferentially under inflammatory conditions. J Immunol. 2009;183:2142–2149. doi: 10.4049/jimmunol.0901059. [DOI] [PubMed] [Google Scholar]
  • 34.Coquet JM, Chakravarti S, Kyparissoudis K, McNab FW, Pitt LA, McKenzie BS, Berzins SP, Smyth MJ, Godfrey DI. Diverse cytokine production by NKT cell subsets and identification of an IL-17-producing CD4-NK1. 1- NKT cell population. Proc Natl Acad Sci U S A. 2008;105:11287–11292. doi: 10.1073/pnas.0801631105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Michel ML, Keller AC, Paget C, Fujio M, Trottein F, Savage PB, Wong CH, Schneider E, Dy M, Leite-de-Moraes MC. Identification of an IL-17-producing NK1. 1(neg) iNKT cell population involved in airway neutrophilia. J Exp Med. 2007;204:995–1001. doi: 10.1084/jem.20061551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Matsuda JL, Gapin L, Sidobre S, Kieper WC, Tan JT, Ceredig R, Surh CD, Kronenberg M. Homeostasis of V alpha 14i NKT cells. Nat Immunol. 2002;3:966–974. doi: 10.1038/ni837. [DOI] [PubMed] [Google Scholar]
  • 37.Milpied P, Massot B, Renand A, Diem S, Herbelin A, Leite-de-Moraes M, Rubio MT, Hermine O. IL-17-producing invariant NKT cells in lymphoid organs are recent thymic emigrants identified by neuropilin-1 expression. Blood. 2011;118:2993–3002. doi: 10.1182/blood-2011-01-329268. [DOI] [PubMed] [Google Scholar]
  • 38.Townsend MJ, Weinmann AS, Matsuda JL, Salomon R, Farnham PJ, Biron CA, Gapin L, Glimcher LH. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity. 2004;20:477–494. doi: 10.1016/s1074-7613(04)00076-7. [DOI] [PubMed] [Google Scholar]
  • 39.van Gisbergen KP, Kragten NA, Hertoghs KM, Wensveen FM, Jonjic S, Hamann J, Nolte MA, van Lier RA. Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation. Nat Immunol. 2012;13:864–871. doi: 10.1038/ni.2393. [DOI] [PubMed] [Google Scholar]
  • 40.Bezbradica JS, Hill T, Stanic AK, Van Kaer L, Joyce S. Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny. Proc Natl Acad Sci U S A. 2005;102:5114–5119. doi: 10.1073/pnas.0408449102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Guo J, Hawwari A, Li H, Sun Z, Mahanta SK, Littman DR, Krangel MS, He YW. Regulation of the TCRalpha repertoire by the survival window of CD4(+)CD8(+) thymocytes. Nat Immunol. 2002;3:469–476. doi: 10.1038/ni791. [DOI] [PubMed] [Google Scholar]
  • 42.Kim PJ, Pai SY, Brigl M, Besra GS, Gumperz J, Ho IC. GATA-3 regulates the development and function of invariant NKT cells. J Immunol. 2006;177:6650–6659. doi: 10.4049/jimmunol.177.10.6650. [DOI] [PubMed] [Google Scholar]
  • 43.Wang L, Carr T, Xiong Y, Wildt KF, Zhu J, Feigenbaum L, Bendelac A, Bosselut R. The sequential activity of Gata3 and Thpok is required for the differentiation of CD1d-restricted CD4+ NKT cells. Eur J Immunol. 2010;40:2385–2390. doi: 10.1002/eji.201040534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Laslo P, Spooner CJ, Warmflash A, Lancki DW, Lee HJ, Sciammas R, Gantner BN, Dinner AR, Singh H. Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell. 2006;126:755–766. doi: 10.1016/j.cell.2006.06.052. [DOI] [PubMed] [Google Scholar]
  • 45.Kaplan MH, Wurster AL, Smiley ST, Grusby MJ. Stat6-dependent and -independent pathways for IL-4 production. J Immunol. 1999;163:6536–6540. [PubMed] [Google Scholar]
  • 46.Stetson DB, Mohrs M, Reinhardt RL, Baron JL, Wang ZE, Gapin L, Kronenberg M, Locksley RM. Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J Exp Med. 2003;198:1069–1076. doi: 10.1084/jem.20030630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Weinreich MA, Odumade OA, Jameson SC, Hogquist KA. T cells expressing the transcription factor PLZF regulate the development of memory-like CD8+ T cells. Nat Immunol. 2010;11:709–716. doi: 10.1038/ni.1898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Weinreich MA, Takada K, Skon C, Reiner SL, Jameson SC, Hogquist KA. KLF2 transcription-factor deficiency in T cells results in unrestrained cytokine production and upregulation of bystander chemokine receptors. Immunity. 2009;31:122–130. doi: 10.1016/j.immuni.2009.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Odumade OA, Weinreich MA, Jameson SC, Hogquist KA. Kruppel-like factor 2 regulates trafficking and homeostasis of gammadelta T cells. J Immunol. 2010;184:6060–6066. doi: 10.4049/jimmunol.1000511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Atherly LO, Lucas JA, Felices M, Yin CC, Reiner SL, Berg LJ. The tec family tyrosine kinases itk and rlk regulate the development of conventional CD8(+) T cells. Immunity. 2006;25:79–91. doi: 10.1016/j.immuni.2006.05.012. [DOI] [PubMed] [Google Scholar]
  • 51.Broussard C, Fleischecker C, Horai R, Chetana M, Venegas AM, Sharp LL, Hedrick SM, Fowlkes BJ, Schwartzberg PL. Altered development of CD8(+) T cell lineages in mice deficient for the tec kinases itk and rlk. Immunity. 2006;25:93–104. doi: 10.1016/j.immuni.2006.05.011. [DOI] [PubMed] [Google Scholar]
  • 52.Verykokakis M, Boos MD, Bendelac A, Adams EJ, Pereira P, Kee BL. Inhibitor of DNA binding 3 limits development of murine slam-associated adaptor protein-dependent “innate” gammadelta T cells. PLoS One. 2010;5:e9303. doi: 10.1371/journal.pone.0009303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Verykokakis M, Boos MD, Bendelac A, Kee BL. SAP protein-dependent natural killer T-like cells regulate the development of CD8(+) T cells with innate lymphocyte characteristics. Immunity. 2010;33:203–215. doi: 10.1016/j.immuni.2010.07.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Fukuyama T, Kasper LH, Boussouar F, Jeevan T, van Deursen J, Brindle PK. Histone acetyltransferase CBP is vital to demarcate conventional and innate CD8+ T-cell development. Mol Cell Biol. 2009;29:3894–3904. doi: 10.1128/MCB.01598-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Lai D, Zhu J, Wang T, Hu-Li J, Terabe M, Berzofsky JA, Clayberger C, Krensky AM. KLF13 sustains thymic memory-like CD8(+) T cells in BALB/c mice by regulating IL-4-generating invariant natural killer T cells. J Exp Med. 2011;208:1093–1103. doi: 10.1084/jem.20101527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Enders A, Stankovic S, Teh C, Uldrich AP, Yabas M, Juelich T, Altin JA, Frankenreiter S, Bergmann H, Roots CM, et al. ZBTB7B (Th-POK) Regulates the Development of IL-17-Producing CD1d-Restricted Mouse NKT Cells. J Immunol. 2012 doi: 10.4049/jimmunol.1201486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Engel I, Zhao M, Kappes D, Taniuchi I, Kronenberg M. The transcription factor Th-POK negatively regulates Th17 differentiation in Vα14i NKT cells. Blood. 2012 doi: 10.1182/blood-2012-01-406280. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Chang PP, Barral P, Fitch J, Pratama A, Ma CS, Kallies A, Hogan JJ, Cerundolo V, Tangye SG, Bittman R, et al. Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat Immunol. 2012;13:35–43. doi: 10.1038/ni.2166. [DOI] [PubMed] [Google Scholar]
  • 59.King IL, Fortier A, Tighe M, Dibble J, Watts GF, Veerapen N, Haberman AM, Besra GS, Mohrs M, Brenner MB, et al. Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner. Nat Immunol. 2012;13:44–50. doi: 10.1038/ni.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Monteiro M, Almeida CF, Caridade M, Ribot JC, Duarte J, Agua-Doce A, Wollenberg I, Silva-Santos B, Graca L. Identification of regulatory Foxp3+ invariant NKT cells induced by TGF-beta. J Immunol. 2010;185:2157–2163. doi: 10.4049/jimmunol.1000359. [DOI] [PubMed] [Google Scholar]
  • 61.Moreira-Teixeira L, Resende M, Devergne O, Herbeuval JP, Hermine O, Schneider E, Dy M, Cordeiro-da-Silva A, Leite-de-Moraes MC. Rapamycin combined with TGF-beta converts human invariant NKT cells into suppressive Foxp3+ regulatory cells. J Immunol. 2012;188:624–631. doi: 10.4049/jimmunol.1102281. [DOI] [PubMed] [Google Scholar]
  • 62.Finlay DK, Kelly AP, Clarke R, Sinclair LV, Deak M, Alessi DR, Cantrell DA. Temporal differences in the dependency on phosphoinositide-dependent kinase 1 distinguish the development of invariant Valpha14 NKT cells and conventional T cells. J Immunol. 2010;185:5973–5982. doi: 10.4049/jimmunol.1000827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Dose M, Sleckman BP, Han J, Bredemeyer AL, Bendelac A, Gounari F. Intrathymic proliferation wave essential for Valpha14+ natural killer T cell development depends on c-Myc. Proc Natl Acad Sci U S A. 2009;106:8641–8646. doi: 10.1073/pnas.0812255106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Mycko MP, Ferrero I, Wilson A, Jiang W, Bianchi T, Trumpp A, MacDonald HR. Selective requirement for c-Myc at an early stage of V(alpha)14i NKT cell development. J Immunol. 2009;182:4641–4648. doi: 10.4049/jimmunol.0803394. [DOI] [PubMed] [Google Scholar]
  • 65.Yang K, Neale G, Green DR, He W, Chi H. The tumor suppressor Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function. Nat Immunol. 2011;12:888–897. doi: 10.1038/ni.2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Powell JD, Pollizzi KN, Heikamp EB, Horton MR. Regulation of immune responses by mTOR. Annu Rev Immunol. 2012;30:39–68. doi: 10.1146/annurev-immunol-020711-075024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Allende ML, Zhou D, Kalkofen DN, Benhamed S, Tuymetova G, Borowski C, Bendelac A, Proia RL. S1P1 receptor expression regulates emergence of NKT cells in peripheral tissues. FASEB J. 2008;22:307–315. doi: 10.1096/fj.07-9087com. [DOI] [PubMed] [Google Scholar]

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