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ScienceDirect Invariant NKT cell development: focus on NOD mice Liana Ghazarian1,2, Yannick Simoni1,2, Isabelle Magalhaes1,2 and Agne`s Lehuen1,2 Natural killer T (NKT) cells are non-conventional T lymphocytes expressing a TCRab and several NK cell markers. Once activated, they can rapidly secrete large amounts of cytokines such as IFN-g and IL-4. As a result they can favor both Th1 and Th2 immune responses and play a critical role in antipathogenic immune responses as well as in regulation of autoimmune diseases. It has now been clearly established that iNKT cells can be subdivided into three subpopulations: iNKT1, iNKT2 and iNKT17 cells. Each of these populations is characterized by the expression of a particular transcription factor, surface markers and cytokines making them functionally distinct. Interestingly, NOD mice developing autoimmune diabetes exhibit a high frequency of iNKT17 cells, which can participate in the disease. Addresses 1 INSERM, U1016, Hospital Cochin/St Vincent de Paul, Paris, France 2 Universite´ Paris Descartes, Laboratoire d’Excellence INFLAMEX, Sorbonne Paris Cite´, Paris, France Corresponding author: Lehuen, Agne`s ([email protected])
Current Opinion in Immunology 2014, 27:83–88 This review comes from a themed issue on Lymphocyte development Edited by Gary W Litman and Claudia Mauri
0952-7915/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coi.2014.02.004
Introduction Natural killer T (NKT) cells are non-conventional T cells expressing a TCRab and several NK cell markers [1]. NKT cells are restricted to the non-polymorphic MHClike molecule CD1d, which presents glycolipids [2]. They are considered to be innate like cells due to their activatory/effector phenotype and are highly conserved in humans and in mice [3]. Two major types of NKT cells are defined: type I or invariant NKT (iNKT) cells and type II NKT cells. This review will focus on the development of iNKT cells and will highlight the role of different iNKT cell subsets in NOD mice.
iNKT cell development Murine iNKT cells express Va14-Ja18 rearranged T cell receptor (TCR) a chain that binds to Vb2, 7 or 8.2 chains in mice (Va24-Ja18 chain pairs with a single Vb11 chain in humans). They are detected by CD1d tetramers loaded with an agonist, the glycolipid aGalactosylCeramide www.sciencedirect.com
(aGalCer), which was originally isolated from a marine sponge. Using this tool, several groups have determined that iNKT cells develop in the thymus after birth since iNKT cells are absent in nude or thymectomised mice [4,5]. iNKT cells develop from the common lymphoid progenitor from which CD4 and CD8 T lymphocytes originate. At the double positive (DP) stage, the transcription factor retinoic acid receptor-related orphan receptor g (RORgt) indirectly induces the expression of anti-apoptotic BCL-xL and prolongs the survival of thymocytes allowing the rearrangement of distal Va14 and Ja18 segments of TCRa chain [6]. After the effective rearrangement of TCRab chains, DP thymocytes encounter classical MHC class I, class II molecules expressed on thymic epithelial cells or CD1d expressed by cortical DP thymocytes [7]. Thymocytes that recognize CD1d become NKT cells. Among this CD1drestricted NKT cell population, cells with a Va14-Ja18 rearranged TCR form the iNKT cell pool. After the DP stage, CD8 is downregulated in mice (CD8 can be expressed by human iNKT cells [8]) and iNKT cells engage in the maturation process which, on the basis of the expression of different markers, has been classically divided into four stages [9]. Stage 0 murine CD44 NK1.1 CD4+ iNKT cells rapidly enter stage 1 where they actively proliferate and enter stage 2 at which point they upregulate CD44 and can downregulate or not CD4. Both CD4+ and CD4 iNKT cells can progress to stage 3 in the thymus where they upregulate NK1.1+. In the periphery, however, most stage 3 NK1.1+ iNKT cells are cells that have left the thymus at stage 2 of development and then progressed to stage 3 by upregulating NK1.1 [4,5,10]. The interaction between DP thymocytes and iNKT cells involves SLAM receptors that signal through (SLAM)-associated protein (SAP) and the kinase Fyn and both Fyn / as well as SAP / mice have a reduced frequency of iNKT cells and defects in their maturation [11–14]. In the thymus and cord blood of human neonates around 90% of iNKT cells express CD4. This number decreases to 40% in the peripheral blood of adults [15]. It is not yet known whether CD4 iNKT cells found in adult peripheral blood are iNKT cells newly emerged from the thymus, and whether they correspond to expanded CD4 iNKT cells or to iNKT cells that have downregulated the CD4 expression.
iNKT cell subpopulations Similar to Th1/Th2/Th17 classification of CD4 T lymphocytes, iNKT cells are suggested to form Current Opinion in Immunology 2014, 27:83–88
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iNKT1/iNKT2/iNKT17 subsets based on their surface markers and particularly on their cytokine production [16]. aGalCer stimulated murine iNKT1 cells produce higher levels of IFN-g than IL-4 and are suggested to be at stage 3 of maturation since they express NK1.1 [5]. By contrast, murine iNKT2 cells mainly produce IL-4 and are found at stage 1 and 2 of maturation forming a finally differentiated population that will not progress to stage 3 and give rise to NKT1 cells [5,17]. iNKT2 cells are characterized by the expression of CD4 and lack of NK1.1 expression. Finally, murine iNKT17 cells produce cytokines typically associated with Th17 lymphocytes such as IL-17 and IL-22 [18]. Thymic iNKT17 cells arise at stage 2 of thymic maturation at which point iNKT cells can be iNKT cells and divided into RORgt+CD4 RORgt CD4+ iNKT cells [19,20]. iNKT cells that retain their expression of RORgt become IL-17 producing iNKT17 cells the majority of which are characterized by the expression of CCR6, CD103 and CD121 (IL-1R) and the lack of expression of CD4 or NK1.1. Whether iNKT17 cells can arise in the periphery is currently debated. Using TGF-b and IL-1b cytokines, iNKT17 cells could be induced from murine splenic or liver RORgt CD4+/ NK1.1+/ iNKT cells in the study by Monteiro et al. [21,22], but these results were not confirmed in the study by Michel et al. [19,23]. iNKT17 cells seem to have a high turnover, since in the periphery they express neuropilin-1 that characterizes recent thymic immigrants [24]. The frequency of iNKT17 cells varies in different organs. They form a small fraction of total iNKT cells in the spleen and liver, but are predominant in the skin and peripheral lymph nodes of C57BL/6 mice [25]. iNKT17 cells have not been detected in human peripheral blood cells stimulated with a nonspecific mitogen, but can be generated from human peripheral blood by using TGF-b, IL-1b and IL-23 cytokines suggesting that these cells can perhaps be present in inflammatory settings [26,27]. The comparison of human and murine iNKT cells shows strong similarities. In the peripheral blood IFN-g and TNF-a are produced by both CD4 and CD4+ iNKT cells which, therefore, resemble murine CD4 and CD4+ iNKT1 cells [8]. By contrast Th2 cytokines are almost exclusively produced by CD4+ iNKT cells, therefore, resembling iNKT2 cells [28]. Interestingly, the frequency of CD161 expressing iNKT cells (CD161 being the human homologue of NK1.1) increases in the periphery of adults compared to the frequency of neonatal thymus suggesting that perhaps similar to mice iNKT cells emerge from the thymus at stage 2 of development and progress to stage 3 by upregulating CD161 in the periphery [15]. However, the studies concerning the developmental stages of human iNKT cells in the thymus remain scarce due to a limited access to neonatal thymic tissue. Current Opinion in Immunology 2014, 27:83–88
Main transcription factors responsible for differentiation of iNKT cell subsets While some transcription factors such as promyelocytic leukemia zinc finger (PLZF) are critical for the development of iNKT cells [29,30], others are necessary for shaping iNKT cell subtypes. Similar to Th1, Th2 and Th17 CD4 T lymphocytes, iNKT cell subtypes are regulated by the transcription factors T-bet, GATA-3 and RORgt respectively.iNKT1 cells are characterized by high expression of T-bet. The numbers of iNKT1 cells are strongly decreased in T-bet / mice in which most iNKT cells remain at stage 2 of development promoting the generation of iNKT2 and iNKT17 cells [31]. iNKT2 cell maturation greatly depends on GATA-3 since in GATA-3 deficient mice the number of thymic and peripheral iNKT2 is greatly reduced while the number of iNKT1 cells is strongly increased [32]. In the periphery of GATA3 / mice iNKT cells produce neither IFN-g nor IL-4 after aGalCer stimulation but can produce IFN-g after PMA/ ionomycine stimulation showing the importance of GATA3 for the production of Th2 type cytokines by iNKT2 cells. The expression of RORgt and, by consequence, the generation of iNKT17 cells is negatively regulated by the transcription factor T helper Poxviruses and zincfinger and Kru¨ppel family (Th-POK) [33]. Mice deficient in Th-POK have an increased expression of genes associated with Th17 lineage, such as RORgt, CD103 and CD121 and have an increased number of IL-17 producing iNKT cells in the periphery.
Signals favoring the differentiation of iNKT cell subtypes It is not yet known as what drives the differentiation of different iNKT cell subsets. The role of CD1d in NKT cell development is critical since mice lacking CD1d expression in the thymus are deficient in NKT cells. However, the sole presence of CD1d is not enough; CD1d must be loaded with lipid antigens in late endosomal/lysosomal compartments and must generate a TCR signaling (Fig. 1) [34,35]. Accordingly, the numbers of iNKT cells are strongly decreased in mice with different types of lysosomal storage disease in which CD1d remains unloaded [34]. Also, patients diagnosed with adrenoleukodystrophy, characterized by an accumulation of saturated very long chain fatty acids, have a lower frequency of iNKT cells compared to control subjects [36]. Recently Facciotti et al. showed that Gnpat / mice, deficient in peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT) that is necessary for the production of ether lipids have significantly lower number of stage 1 and 2 and particularly stage 3 iNKT cell both in the thymus and in the periphery compared to wild type mice [37]. Thus, lipid antigens can perhaps favor a certain type of iNKT cells. www.sciencedirect.com
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Figure 1
(2) A particular IL-15
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Different external signals could regulate the differentiation of various iNKT cell subsets. (1) iNKT1, iNKT2 and iNKT17 cell express the receptors for IL-15 (IL-2Rb), IL-25 (IL-17RB) and IL-23 (IL-23R) cytokines respectively which can, therefore, favor their differentiation; (2) apart from cytokines, the nature of self-antigen presented by CD1d might favor one subset of iNKT cell over another; (3) finally, depending on the configuration of CD1d:ligand complex with TCR, the intracellular signaling in iNKT cells can by of varying strength thus potentially influencing the proportions of different iNKT cell populations.
A weak TCR signal favors the differentiation of naı¨ve CD4 T lymphocytes into Th2 cells. iNKT cells, while all having the Va14-Ja18 rearrangement, can have different affinities for the CD1d:ligand complex due to hypermutations in CDR2b and CDR3b loops [38]. Besides, Lee et al. observed that a higher frequency of iNKT2 cells used Vb2 and Vb7 chains compared to iNKT1 and iNKT17 subgroups [17]. It is perhaps possible that similar to CD4 T lymphocytes, the strengths of the signal from interactions of iNKT cell TCR with the CD1d:ligand complex can induce the upregulation of a particular transcription factor and favor a specific iNKT cell subtype.
frequency of different iNKT cell subsets correlates with the type of immune response generated in these mice. Thus, in the thymus iNKT1 cells are more abundant C57BL/6 mice which often mount Th1 immune response, while iNKT2 cells are more abundant in BALB/c mice compared to C57BL/6 mice (Fig. 2) [17]. The frequency of iNKT2 cells is also strongly increased in the lung of BALB/c mice in which they contribute to viral induced airway hyperreactivity and asthma since CD1d / BALB/c mice which are devoid of iNKT cells are resistant to asthma [39,41,42]. In C57BL/6 mice iNKT1 cells are mostly found in the liver and spleen.
Besides TCR signaling, cytokines can play an important role in the generation of iNKT cell subsets. Watarai et al. have shown that IL-17RB, receptor of IL-25, plays an important role in the generation of iNKT2 and iNKT17 cells. Thus IL-17RB+CD4+ iNKT cells mainly produce Th2 type cytokines IL-4, IL-10 and IL-13 specific to iNKT2 cells, IL-17RB+CD4 iNKT cells mainly produce IL-17, IL-22 and express RORgt specific to iNKT17 cells [39]. While iNKT1 cells do not depend on IL-17RB, they do require IL-15 for the upregulation of NK1.1 since mice having a mutated IL-15, besides having decreased numbers of iNKT cells, lack NK1.1 expressing iNKT1 cells [39]. Moreover, mice deficient in the transcription factor early growth response protein 2 (egr2), which induces the expression of IL-2R b-chain necessary for the formation of IL-15 signaling receptor, fail to upregulate NK1.1 and have iNKT1 cells [40].
Nonobese diabetic (NOD) mice develop spontaneous autoimmune type 1 diabetes (T1D) characterized by the destruction of pancreatic islet beta cells. NOD mice have lower frequency of iNKT cells compared to non-autoimmune strains suggesting that they play a protective role in this pathology [43,44]. Moreover, CD1d / NOD mice develop an accelerated T1D compared to control NOD mice. Conversely, increasing iNKT cell number effectively prevents T1D development [45,46]. Besides numerical differences, iNKT cells of NOD mice also have defects in the production of protective Th2 cytokines IL-4 and IL-10 compared to C57BL/6 mice [44,47]. However, the low IL-4 secretion by iNKT cells is due to impaired interaction of SLAM receptors on iNKT2 cells and antigen presenting cells rather than decreased iNKT2 cell generation in the thymus since iNKT2 cell frequency is higher in NOD mice compared to C57BL/6 mice [48,49].
iNKT cells in NOD mice
Besides defect in Th2 type response, the comparison of iNKT cell subsets revealed that, contrary to the nonautoimmune C57BL/6 mice, NOD mice have an elevated
Mouse models used in laboratories differ in the type of immune response they mount. Interestingly, the www.sciencedirect.com
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Figure 2
Thymus
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Murine strains differ in the proportions of different iNKT cell subsets. The frequency of iNKT1 cells is the highest in the thymus of C57BL/6 mice compared to BALB/c and NOD mice. By contrast, iNKT2 and iNKT17 cells predominate in the thymus of BALB/c mice. Lastly, NOD mice have intermediate levels of these populations compared to both C57BL/6 and BALB/c mice.
number and frequency of iNKT17 cells in the thymus [20]. Interestingly, non-autoimmune BALB/c mice have an even higher frequency of iNKT17 cells in the thymus. However, in the periphery the frequency of iNKT17 cells of BALB/c mice is much lower compared to NOD mice (Simoni and Lehuen, unpublished data). iNKT17 cells can be found in the pancreas of prediabetic NOD mice, while these and other immune cells are virtually absent in the pancreas of non-autoimmune strains. Importantly, the analysis of IL-17 mRNA in iNKT17 cells of various tissues of NOD mice revealed that iNKT cells express high levels of IL-17 transcript specifically in the pancreas compared to other peripheral tissues such as mesenteric or inguinal lymph nodes [20]. In diabetes transfer experiments, iNKT17 cells exacerbate the development of T1D and increase the incidence of diabetes of NOD mice through IL-17 secretion. Moreover, repetitive aGalCer injections that have been shown to protect NOD mice from developing diabetes, strongly decrease the production of both IFN-g and IL-17 by iNKT cells suggesting a deleterious role for IL-17 in spontaneous diabetes development of NOD mice. It remains to be identified whether iNKT17 cells are present in the pancreas of diabetic subjects. Overall, the comparison of different murine strains reveals that the frequency of iNKT cell subpopulations in the thymus does not necessarily correlate with their Current Opinion in Immunology 2014, 27:83–88
frequency in the periphery. It will therefore be interesting to determine whether these differences are due to the poor survival or thymic export from the periphery.
Conclusion iNKT cells are potent regulators of immune responses against pathogens, autoimmunity or malignancy despite their relatively low frequency compared to other lymphocytes. The curious capacity of iNKT cells to produce both Th1 and Th2 type cytokines and promote both Th1 and Th2 type immune responses depending to the pathological environment has kept them at the interest of the scientific community. It is becoming clearer that iNKT cells do not form a homogenous cell population, but rather form phenotypically and functionally distinct subpopulations. Each of these populations can favor a certain type of immune response that can be beneficial or, on the contrary, deleterious for the organism. New studies are therefore critical as they can help identifying how to orient iNKT cells towards a desired phenotype and favor the most appropriate immune response. Interestingly in human decreased circulating iNKT cell numbers have been reported in a wide varieties of autoimmune disorders including systemic lupus erythematosus and multiple sclerosis [50–52]. However in T1D patients the difference in iNKT cell frequency remains controversial [53]. iNKT cells are also affected in their ability to produced cytokines in some autoimmune diseases. Defects in IL-4 production by www.sciencedirect.com
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iNKT cells have been observed in pancreatic lymph nodes of T1D patients, whereas IL-4 production by iNKT cells is increased in circulating iNKT cells in multiple sclerosis patients upon remission [54,55]. In systemic lupus erythematosus patients, the production of IL-2, IFN-g and TNFa, but not IL-4, by iNKT cells is dramatically reduced due to abnormal CD1d presentation by B cells in these patients [51]. Of note, these differences in cytokine production by iNKT cells in human autoimmune disorders seem to reflect the activation status of iNKT cells in the periphery and the role of iNKT cell subpopulation development remains an open and important question.
12. Chung B, Aoukaty A, Dutz J, Terhorst C, Tan R: Signaling lymphocytic activation molecule-associated protein controls NKT cell functions. J Immunol 2005, 174:3153-3157.
Acknowledgements
16. Brennan PJ, Brigl M, Brenner MB: Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol 2013, 13:101-117.
This work was supported by funds from the Institut National de la Sante´ et de la Recherche Me´dicale and the Centre National pour la Recherche Scientifique, grant from ANR-09-GENO-023 and Laboratoire d’Excellence INFLAMEX to Agne`s Lehuen who is also a recipient of an APHP-CNRS Contrat Hospitalier de Recherche Translationnelle.
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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. 42. Akbari O, Stock P, Meyer E, Kronenberg M, Sidobre S, Nakayama T, Taniguchi M, Grusby MJ, DeKruyff RH, Umetsu DT: Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nat Med 2003, 9:582-588. The publication by Terashima et al. allowed to identify a new population of iNKT cells expressing IL-25 receptor B. These cells produce Th2 type cytokines in response to IL-25 stimulation and induce airway hypersensitive reaction upon in vivo IL-25 treatment. 43. Hammond KJ, Pellicci DG, Poulton LD, Naidenko OV, Scalzo AA, Baxter AG, Godfrey DI: CD1d-restricted NKT cells: an interstrain comparison. J Immunol 2001, 167:1164-1173. 44. Gombert JM, Herbelin A, Tancrede-Bohin E, Dy M, Carnaud C, Bach JF: Early quantitative and functional deficiency of NK1+like thymocytes in the NOD mouse. Eur J Immunol 1996, 26:2989-2998. 45. Lehuen A, Lantz O, Beaudoin L, Laloux V, Carnaud C, Bendelac A, Bach JF, Monteiro RC: Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes. J Exp Med 1998, 188:1831-1839. Refs. [45,46] show the capacity of iNKT cells to prevent type 1 diabetes in autoimmune diabetes prone NOD mice. 46. Hammond KJ, Poulton LD, Palmisano LJ, Silveira PA, Godfrey DI, Baxter AG: Alpha/beta-T cell receptor (TCR)+CD4SCD8S (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J Exp Med 1998, 187:1047-1056. See annotation to Ref. [45]. 47. Laloux V, Beaudoin L, Jeske D, Carnaud C, Lehuen A: NK T cell-induced protection against diabetes in V alpha 14-J alpha 281 transgenic nonobese diabetic mice is associated with a Th2 shift circumscribed regionally to the islets and functionally to islet autoantigen. J Immunol 2001, 166:37493756. 48. Baev DV, Caielli S, Ronchi F, Coccia M, Facciotti F, Nichols KE, Falcone M: Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice. J Immunol 2008, 181:869-877. 49. Jordan MA, Fletcher JM, Pellicci D, Baxter AG: Slamf1, the NKT cell control gene Nkt1. J Immunol 2007, 178:1618-1627. 50. van der Vliet HJ, von Blomberg BM, Nishi N, Reijm M, Voskuyl AE, van Bodegraven AA, Polman CH, Rustemeyer T, Lips P, van den Eertwegh AJ et al.: Circulating V(alpha24+) Vbeta11+ NKT cell numbers are decreased in a wide variety of diseases that are characterized by autoreactive tissue damage. Clin Immunol 2001, 100:144-148. 51. Bosma A, Abdel-Gadir A, Isenberg DA, Jury EC, Mauri C: Lipidantigen presentation by CD1d(+) B cells is essential for the maintenance of invariant natural killer T cells. Immunity 2012, 36:477-490. 52. Illes Z, Kondo T, Newcombe J, Oka N, Tabira T, Yamamura T: Differential expression of NK T cell V alpha 24J alpha Q invariant TCR chain in the lesions of multiple sclerosis and chronic inflammatory demyelinating polyneuropathy. J Immunol 2000, 164:4375-4381. 53. Lehuen A, Diana J, Zaccone P, Cooke A: Immune cell crosstalk in type 1 diabetes. Nat Rev Immunol 2010, 10:501-513. 54. Kent SC, Chen Y, Clemmings SM, Viglietta V, Kenyon NS, Ricordi C, Hering B, Hafler DA: Loss of IL-4 secretion from human type 1a diabetic pancreatic draining lymph node NKT cells. J Immunol 2005, 175:4458-4464. 55. Araki M, Kondo T, Gumperz JE, Brenner MB, Miyake S, Yamamura T: Th2 bias of CD4+ NKT cells derived from multiple sclerosis in remission. Int Immunol 2003, 15:279-288.
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ScienceDirect Invariant NKT cell development: focus on NOD mice Liana Ghazarian1,2, Yannick Simoni1,2, Isabelle Magalhaes1,2 and Agne`s Lehuen1,2 Natural killer T (NKT) cells are non-conventional T lymphocytes expressing a TCRab and several NK cell markers. Once activated, they can rapidly secrete large amounts of cytokines such as IFN-g and IL-4. As a result they can favor both Th1 and Th2 immune responses and play a critical role in antipathogenic immune responses as well as in regulation of autoimmune diseases. It has now been clearly established that iNKT cells can be subdivided into three subpopulations: iNKT1, iNKT2 and iNKT17 cells. Each of these populations is characterized by the expression of a particular transcription factor, surface markers and cytokines making them functionally distinct. Interestingly, NOD mice developing autoimmune diabetes exhibit a high frequency of iNKT17 cells, which can participate in the disease. Addresses 1 INSERM, U1016, Hospital Cochin/St Vincent de Paul, Paris, France 2 Universite´ Paris Descartes, Laboratoire d’Excellence INFLAMEX, Sorbonne Paris Cite´, Paris, France Corresponding author: Lehuen, Agne`s ([email protected])
Current Opinion in Immunology 2014, 27:83–88 This review comes from a themed issue on Lymphocyte development Edited by Gary W Litman and Claudia Mauri
0952-7915/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coi.2014.02.004
Introduction Natural killer T (NKT) cells are non-conventional T cells expressing a TCRab and several NK cell markers [1]. NKT cells are restricted to the non-polymorphic MHClike molecule CD1d, which presents glycolipids [2]. They are considered to be innate like cells due to their activatory/effector phenotype and are highly conserved in humans and in mice [3]. Two major types of NKT cells are defined: type I or invariant NKT (iNKT) cells and type II NKT cells. This review will focus on the development of iNKT cells and will highlight the role of different iNKT cell subsets in NOD mice.
iNKT cell development Murine iNKT cells express Va14-Ja18 rearranged T cell receptor (TCR) a chain that binds to Vb2, 7 or 8.2 chains in mice (Va24-Ja18 chain pairs with a single Vb11 chain in humans). They are detected by CD1d tetramers loaded with an agonist, the glycolipid aGalactosylCeramide www.sciencedirect.com
(aGalCer), which was originally isolated from a marine sponge. Using this tool, several groups have determined that iNKT cells develop in the thymus after birth since iNKT cells are absent in nude or thymectomised mice [4,5]. iNKT cells develop from the common lymphoid progenitor from which CD4 and CD8 T lymphocytes originate. At the double positive (DP) stage, the transcription factor retinoic acid receptor-related orphan receptor g (RORgt) indirectly induces the expression of anti-apoptotic BCL-xL and prolongs the survival of thymocytes allowing the rearrangement of distal Va14 and Ja18 segments of TCRa chain [6]. After the effective rearrangement of TCRab chains, DP thymocytes encounter classical MHC class I, class II molecules expressed on thymic epithelial cells or CD1d expressed by cortical DP thymocytes [7]. Thymocytes that recognize CD1d become NKT cells. Among this CD1drestricted NKT cell population, cells with a Va14-Ja18 rearranged TCR form the iNKT cell pool. After the DP stage, CD8 is downregulated in mice (CD8 can be expressed by human iNKT cells [8]) and iNKT cells engage in the maturation process which, on the basis of the expression of different markers, has been classically divided into four stages [9]. Stage 0 murine CD44 NK1.1 CD4+ iNKT cells rapidly enter stage 1 where they actively proliferate and enter stage 2 at which point they upregulate CD44 and can downregulate or not CD4. Both CD4+ and CD4 iNKT cells can progress to stage 3 in the thymus where they upregulate NK1.1+. In the periphery, however, most stage 3 NK1.1+ iNKT cells are cells that have left the thymus at stage 2 of development and then progressed to stage 3 by upregulating NK1.1 [4,5,10]. The interaction between DP thymocytes and iNKT cells involves SLAM receptors that signal through (SLAM)-associated protein (SAP) and the kinase Fyn and both Fyn / as well as SAP / mice have a reduced frequency of iNKT cells and defects in their maturation [11–14]. In the thymus and cord blood of human neonates around 90% of iNKT cells express CD4. This number decreases to 40% in the peripheral blood of adults [15]. It is not yet known whether CD4 iNKT cells found in adult peripheral blood are iNKT cells newly emerged from the thymus, and whether they correspond to expanded CD4 iNKT cells or to iNKT cells that have downregulated the CD4 expression.
iNKT cell subpopulations Similar to Th1/Th2/Th17 classification of CD4 T lymphocytes, iNKT cells are suggested to form Current Opinion in Immunology 2014, 27:83–88
84 Lymphocyte development
iNKT1/iNKT2/iNKT17 subsets based on their surface markers and particularly on their cytokine production [16]. aGalCer stimulated murine iNKT1 cells produce higher levels of IFN-g than IL-4 and are suggested to be at stage 3 of maturation since they express NK1.1 [5]. By contrast, murine iNKT2 cells mainly produce IL-4 and are found at stage 1 and 2 of maturation forming a finally differentiated population that will not progress to stage 3 and give rise to NKT1 cells [5,17]. iNKT2 cells are characterized by the expression of CD4 and lack of NK1.1 expression. Finally, murine iNKT17 cells produce cytokines typically associated with Th17 lymphocytes such as IL-17 and IL-22 [18]. Thymic iNKT17 cells arise at stage 2 of thymic maturation at which point iNKT cells can be iNKT cells and divided into RORgt+CD4 RORgt CD4+ iNKT cells [19,20]. iNKT cells that retain their expression of RORgt become IL-17 producing iNKT17 cells the majority of which are characterized by the expression of CCR6, CD103 and CD121 (IL-1R) and the lack of expression of CD4 or NK1.1. Whether iNKT17 cells can arise in the periphery is currently debated. Using TGF-b and IL-1b cytokines, iNKT17 cells could be induced from murine splenic or liver RORgt CD4+/ NK1.1+/ iNKT cells in the study by Monteiro et al. [21,22], but these results were not confirmed in the study by Michel et al. [19,23]. iNKT17 cells seem to have a high turnover, since in the periphery they express neuropilin-1 that characterizes recent thymic immigrants [24]. The frequency of iNKT17 cells varies in different organs. They form a small fraction of total iNKT cells in the spleen and liver, but are predominant in the skin and peripheral lymph nodes of C57BL/6 mice [25]. iNKT17 cells have not been detected in human peripheral blood cells stimulated with a nonspecific mitogen, but can be generated from human peripheral blood by using TGF-b, IL-1b and IL-23 cytokines suggesting that these cells can perhaps be present in inflammatory settings [26,27]. The comparison of human and murine iNKT cells shows strong similarities. In the peripheral blood IFN-g and TNF-a are produced by both CD4 and CD4+ iNKT cells which, therefore, resemble murine CD4 and CD4+ iNKT1 cells [8]. By contrast Th2 cytokines are almost exclusively produced by CD4+ iNKT cells, therefore, resembling iNKT2 cells [28]. Interestingly, the frequency of CD161 expressing iNKT cells (CD161 being the human homologue of NK1.1) increases in the periphery of adults compared to the frequency of neonatal thymus suggesting that perhaps similar to mice iNKT cells emerge from the thymus at stage 2 of development and progress to stage 3 by upregulating CD161 in the periphery [15]. However, the studies concerning the developmental stages of human iNKT cells in the thymus remain scarce due to a limited access to neonatal thymic tissue. Current Opinion in Immunology 2014, 27:83–88
Main transcription factors responsible for differentiation of iNKT cell subsets While some transcription factors such as promyelocytic leukemia zinc finger (PLZF) are critical for the development of iNKT cells [29,30], others are necessary for shaping iNKT cell subtypes. Similar to Th1, Th2 and Th17 CD4 T lymphocytes, iNKT cell subtypes are regulated by the transcription factors T-bet, GATA-3 and RORgt respectively.iNKT1 cells are characterized by high expression of T-bet. The numbers of iNKT1 cells are strongly decreased in T-bet / mice in which most iNKT cells remain at stage 2 of development promoting the generation of iNKT2 and iNKT17 cells [31]. iNKT2 cell maturation greatly depends on GATA-3 since in GATA-3 deficient mice the number of thymic and peripheral iNKT2 is greatly reduced while the number of iNKT1 cells is strongly increased [32]. In the periphery of GATA3 / mice iNKT cells produce neither IFN-g nor IL-4 after aGalCer stimulation but can produce IFN-g after PMA/ ionomycine stimulation showing the importance of GATA3 for the production of Th2 type cytokines by iNKT2 cells. The expression of RORgt and, by consequence, the generation of iNKT17 cells is negatively regulated by the transcription factor T helper Poxviruses and zincfinger and Kru¨ppel family (Th-POK) [33]. Mice deficient in Th-POK have an increased expression of genes associated with Th17 lineage, such as RORgt, CD103 and CD121 and have an increased number of IL-17 producing iNKT cells in the periphery.
Signals favoring the differentiation of iNKT cell subtypes It is not yet known as what drives the differentiation of different iNKT cell subsets. The role of CD1d in NKT cell development is critical since mice lacking CD1d expression in the thymus are deficient in NKT cells. However, the sole presence of CD1d is not enough; CD1d must be loaded with lipid antigens in late endosomal/lysosomal compartments and must generate a TCR signaling (Fig. 1) [34,35]. Accordingly, the numbers of iNKT cells are strongly decreased in mice with different types of lysosomal storage disease in which CD1d remains unloaded [34]. Also, patients diagnosed with adrenoleukodystrophy, characterized by an accumulation of saturated very long chain fatty acids, have a lower frequency of iNKT cells compared to control subjects [36]. Recently Facciotti et al. showed that Gnpat / mice, deficient in peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT) that is necessary for the production of ether lipids have significantly lower number of stage 1 and 2 and particularly stage 3 iNKT cell both in the thymus and in the periphery compared to wild type mice [37]. Thus, lipid antigens can perhaps favor a certain type of iNKT cells. www.sciencedirect.com
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Figure 1
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Different external signals could regulate the differentiation of various iNKT cell subsets. (1) iNKT1, iNKT2 and iNKT17 cell express the receptors for IL-15 (IL-2Rb), IL-25 (IL-17RB) and IL-23 (IL-23R) cytokines respectively which can, therefore, favor their differentiation; (2) apart from cytokines, the nature of self-antigen presented by CD1d might favor one subset of iNKT cell over another; (3) finally, depending on the configuration of CD1d:ligand complex with TCR, the intracellular signaling in iNKT cells can by of varying strength thus potentially influencing the proportions of different iNKT cell populations.
A weak TCR signal favors the differentiation of naı¨ve CD4 T lymphocytes into Th2 cells. iNKT cells, while all having the Va14-Ja18 rearrangement, can have different affinities for the CD1d:ligand complex due to hypermutations in CDR2b and CDR3b loops [38]. Besides, Lee et al. observed that a higher frequency of iNKT2 cells used Vb2 and Vb7 chains compared to iNKT1 and iNKT17 subgroups [17]. It is perhaps possible that similar to CD4 T lymphocytes, the strengths of the signal from interactions of iNKT cell TCR with the CD1d:ligand complex can induce the upregulation of a particular transcription factor and favor a specific iNKT cell subtype.
frequency of different iNKT cell subsets correlates with the type of immune response generated in these mice. Thus, in the thymus iNKT1 cells are more abundant C57BL/6 mice which often mount Th1 immune response, while iNKT2 cells are more abundant in BALB/c mice compared to C57BL/6 mice (Fig. 2) [17]. The frequency of iNKT2 cells is also strongly increased in the lung of BALB/c mice in which they contribute to viral induced airway hyperreactivity and asthma since CD1d / BALB/c mice which are devoid of iNKT cells are resistant to asthma [39,41,42]. In C57BL/6 mice iNKT1 cells are mostly found in the liver and spleen.
Besides TCR signaling, cytokines can play an important role in the generation of iNKT cell subsets. Watarai et al. have shown that IL-17RB, receptor of IL-25, plays an important role in the generation of iNKT2 and iNKT17 cells. Thus IL-17RB+CD4+ iNKT cells mainly produce Th2 type cytokines IL-4, IL-10 and IL-13 specific to iNKT2 cells, IL-17RB+CD4 iNKT cells mainly produce IL-17, IL-22 and express RORgt specific to iNKT17 cells [39]. While iNKT1 cells do not depend on IL-17RB, they do require IL-15 for the upregulation of NK1.1 since mice having a mutated IL-15, besides having decreased numbers of iNKT cells, lack NK1.1 expressing iNKT1 cells [39]. Moreover, mice deficient in the transcription factor early growth response protein 2 (egr2), which induces the expression of IL-2R b-chain necessary for the formation of IL-15 signaling receptor, fail to upregulate NK1.1 and have iNKT1 cells [40].
Nonobese diabetic (NOD) mice develop spontaneous autoimmune type 1 diabetes (T1D) characterized by the destruction of pancreatic islet beta cells. NOD mice have lower frequency of iNKT cells compared to non-autoimmune strains suggesting that they play a protective role in this pathology [43,44]. Moreover, CD1d / NOD mice develop an accelerated T1D compared to control NOD mice. Conversely, increasing iNKT cell number effectively prevents T1D development [45,46]. Besides numerical differences, iNKT cells of NOD mice also have defects in the production of protective Th2 cytokines IL-4 and IL-10 compared to C57BL/6 mice [44,47]. However, the low IL-4 secretion by iNKT cells is due to impaired interaction of SLAM receptors on iNKT2 cells and antigen presenting cells rather than decreased iNKT2 cell generation in the thymus since iNKT2 cell frequency is higher in NOD mice compared to C57BL/6 mice [48,49].
iNKT cells in NOD mice
Besides defect in Th2 type response, the comparison of iNKT cell subsets revealed that, contrary to the nonautoimmune C57BL/6 mice, NOD mice have an elevated
Mouse models used in laboratories differ in the type of immune response they mount. Interestingly, the www.sciencedirect.com
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86 Lymphocyte development
Figure 2
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Murine strains differ in the proportions of different iNKT cell subsets. The frequency of iNKT1 cells is the highest in the thymus of C57BL/6 mice compared to BALB/c and NOD mice. By contrast, iNKT2 and iNKT17 cells predominate in the thymus of BALB/c mice. Lastly, NOD mice have intermediate levels of these populations compared to both C57BL/6 and BALB/c mice.
number and frequency of iNKT17 cells in the thymus [20]. Interestingly, non-autoimmune BALB/c mice have an even higher frequency of iNKT17 cells in the thymus. However, in the periphery the frequency of iNKT17 cells of BALB/c mice is much lower compared to NOD mice (Simoni and Lehuen, unpublished data). iNKT17 cells can be found in the pancreas of prediabetic NOD mice, while these and other immune cells are virtually absent in the pancreas of non-autoimmune strains. Importantly, the analysis of IL-17 mRNA in iNKT17 cells of various tissues of NOD mice revealed that iNKT cells express high levels of IL-17 transcript specifically in the pancreas compared to other peripheral tissues such as mesenteric or inguinal lymph nodes [20]. In diabetes transfer experiments, iNKT17 cells exacerbate the development of T1D and increase the incidence of diabetes of NOD mice through IL-17 secretion. Moreover, repetitive aGalCer injections that have been shown to protect NOD mice from developing diabetes, strongly decrease the production of both IFN-g and IL-17 by iNKT cells suggesting a deleterious role for IL-17 in spontaneous diabetes development of NOD mice. It remains to be identified whether iNKT17 cells are present in the pancreas of diabetic subjects. Overall, the comparison of different murine strains reveals that the frequency of iNKT cell subpopulations in the thymus does not necessarily correlate with their Current Opinion in Immunology 2014, 27:83–88
frequency in the periphery. It will therefore be interesting to determine whether these differences are due to the poor survival or thymic export from the periphery.
Conclusion iNKT cells are potent regulators of immune responses against pathogens, autoimmunity or malignancy despite their relatively low frequency compared to other lymphocytes. The curious capacity of iNKT cells to produce both Th1 and Th2 type cytokines and promote both Th1 and Th2 type immune responses depending to the pathological environment has kept them at the interest of the scientific community. It is becoming clearer that iNKT cells do not form a homogenous cell population, but rather form phenotypically and functionally distinct subpopulations. Each of these populations can favor a certain type of immune response that can be beneficial or, on the contrary, deleterious for the organism. New studies are therefore critical as they can help identifying how to orient iNKT cells towards a desired phenotype and favor the most appropriate immune response. Interestingly in human decreased circulating iNKT cell numbers have been reported in a wide varieties of autoimmune disorders including systemic lupus erythematosus and multiple sclerosis [50–52]. However in T1D patients the difference in iNKT cell frequency remains controversial [53]. iNKT cells are also affected in their ability to produced cytokines in some autoimmune diseases. Defects in IL-4 production by www.sciencedirect.com
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iNKT cells have been observed in pancreatic lymph nodes of T1D patients, whereas IL-4 production by iNKT cells is increased in circulating iNKT cells in multiple sclerosis patients upon remission [54,55]. In systemic lupus erythematosus patients, the production of IL-2, IFN-g and TNFa, but not IL-4, by iNKT cells is dramatically reduced due to abnormal CD1d presentation by B cells in these patients [51]. Of note, these differences in cytokine production by iNKT cells in human autoimmune disorders seem to reflect the activation status of iNKT cells in the periphery and the role of iNKT cell subpopulation development remains an open and important question.
12. Chung B, Aoukaty A, Dutz J, Terhorst C, Tan R: Signaling lymphocytic activation molecule-associated protein controls NKT cell functions. J Immunol 2005, 174:3153-3157.
Acknowledgements
16. Brennan PJ, Brigl M, Brenner MB: Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat Rev Immunol 2013, 13:101-117.
This work was supported by funds from the Institut National de la Sante´ et de la Recherche Me´dicale and the Centre National pour la Recherche Scientifique, grant from ANR-09-GENO-023 and Laboratoire d’Excellence INFLAMEX to Agne`s Lehuen who is also a recipient of an APHP-CNRS Contrat Hospitalier de Recherche Translationnelle.
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