Immunology and Cell Biology (2009) 87, 361–363 & 2009 Australasian Society for Immunology Inc. All rights reserved 0818-9641/09 $32.00 www.nature.com/icb
NEWS AND COMMENTARY NKT cell development
It’s up to you Egr2 Hamid Bassiri and Kim E Nichols Immunology and Cell Biology (2009) 87, 361–363; doi:10.1038/icb.2009.20; published online 31 March 2009
I
nvariant natural killer T (iNKT) cells comprise a rare subset of innate type lymphocytes with functions in host immunity, including protection from specific pathogens1 and tumors,2 promotion of airway hyperreactivity3 and maintenance of tolerance.4 Given their pleiotropic functions, there has been great interest in elucidating the mechanisms controlling iNKT cell development and activation. In a recent study published in the journal Nature Immunology, Lazarevic et al.5 provide new insights into iNKT cell biology by demonstrating that the gene encoding early growth response 2 (Egr2), a target of the transcription factor nuclear factor of activated T (NFAT) cells, has critical functions during the ontogeny of this unique cell lineage. iNKT cells derive their name from the coexpression of NK cell-specific markers, such as the Ly49 receptors and NK1.1, and the T-cell receptor (TCR). Over 80% of iNKT cells express a canonical or ‘invariant’ TCR, typified by a common TCRa chain rearrangement (Va14-Ja18 in mice; Va24-Ja18 in humans) paired with specific TCRb chains (Vb8, 7, 2 in mice; Vb11 in humans).6 The invariant TCR confers specificity for recognition of glycosphingolipids, such as the prototypical iNKT cell agonist a-galactosylceramide, presented by the CD1 family of molecules on antigen-presenting cells (APC). Engagement of the invariant TCR by CD1– lipid complexes leads to rapid iNKT cell activation, marked by the robust production of TH1- and TH2-type cytokines and upregulation of costimulatory molecules such as CD40L.6 These events contribute to the reciprocal activation of APC, such as dendritic cells and macrophages, as well as the activaH Bassiri is at the Division of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA, and KE Nichols is at the Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA. E-mail:
[email protected]
tion of T and B lymphocytes and NK cells. This multiplicity of functions thus enables iNKT cells to influence the development of both innate and adaptive immune responses. iNKT cells develop in the thymus by undergoing a coordinated series of maturational stages.6 Studies in mice demonstrate that these cells are derived from CD4+CD8+ double-positive (DP) thymocytes, suggesting that until the DP stage, iNKT cell ontogeny is similar to the development of conventional T cells (Figure 1). Upon expression of the invariant TCR, iNKT cell progenitors become susceptible to the effects of selection. Unlike conventional thymocytes, however, which rely on interactions with thymic epithelial cells for delivery of positively selecting signals, iNKT progenitors interact with other CD1d+ DP thymocytes presumably presenting positively selecting lipid self-antigens. The successfully selected cells, which express high levels of CD24 but lack NK1.1 and CD44 (depicted in Figure 1 as Stage 0, according to Savage7), upregulate CD69, begin to proliferate and transit through subsequent stages marked by downregulation of CD24 (Stage 1) and upregulation of CD44 (Stage 2) and eventually NK1.1 (Stage 3). Given their unique developmental and functional characteristics, it has long been proposed that iNKT cells might utilize distinct signaling pathways during ontogeny compared to conventional T cells. Through an elegant set of experiments, Lazarevic et al.5 examined the function of the phosphatase calcineurin and the transcription factor NFAT during iNKT cell development. Engagement of the TCR leads to a series of signals that increase intracellular calcium levels, thereby resulting in the activation of calcineurin, which dephosphorylates cytoplasmic NFAT family members. Dephosphorylated NFAT proteins then translocate to the nucleus, where they regulate expression of specific target genes important for T-cell development and activation. Many studies
have documented that activation of the calcineurin–NFAT pathway is essential for thymocyte development.8 For example, mice lacking expression of the most abundant lymphocyte-specific catalytic subunit of calcineurin (Ab),9 or animals with thymocyte-specific deletion of the regulatory subunit (B1),10 fail to properly dephosphorylate NFAT family members and exhibit marked defects in positive selection. Despite their important function in T-cell selection, it was not previously known whether calcineurin-dependent transcriptional events were also required for iNKT cell development. To address this question, Lazarevic et al.5 used conditional gene deletion to inactivate Cnb1, the gene encoding the regulatory component of calcineurin, in thymocytes. Similar to what was noted in T cells, elimination of calcineurin function also led to significantly reduced percentages and absolute numbers of mature iNKT cells. To understand the mechanisms by which calcineurin–NFAT signaling might influence iNKT cell development, the authors next focused on the Egr family of transcription factors, known targets of NFAT with varied functions in thymocyte development (reviewed by Safford et al.12). After demonstrating that the Egr family members Egr1, Egr2 and Egr3 are each expressed in the thymocytes and splenocytes of wild-type (WT) mice, the authors examined the NKT cell compartment in mice deficient for the expression of these transcription factors in all tissues (Egr1–/–, Egr3–/–) or selectively in hematopoietic cells (Egr2–/– fetal liver chimeras). Surprisingly, absence of Egr2, but not Egr1 or Egr3, led to significant reductions in the percentage and absolute number of iNKT cells, but did not affect development of CD4+ or CD8+ T cells. Egr2-deficient thymocytes exhibited normal expression and function of CD1d, thus ruling out defects in this molecule as a possible cause of aberrant iNKT
News and Commentary 362
CD44loNK1.1−
TCR rearrangement
DP Vα14-Jα18 Vβ8,7 or 2
DN SLAMF
Positive selection SLAM/Ly108 SAP Fyn NFkB
CD4+
CD4+CD8lo CD24hi CD69hi
CD24lo CD69hi
Egr2
1
0
PLZF
SLAMF CD1d + glycolipid DP
hi
CD44 NK1.1
−
CD4+ CD24lo CD122hi IL-4 2
Thymus GATA3 T-bet IL-15
Periphery
TCR Glycolipid CD1d + β2m SLAM family receptor
CD44
Proliferative expansion
hiNK1.1+
CD4+ or CD4−8− CD24loCD122hi IL-4, IFN-γ
3
Figure 1 Thymic invariant natural killer T (iNKT) cell development. iNKT cells develop from CD4+CD8+ double-positive (DP) cells expressing canonical CD1/ glycolipid-reactive T-cell receptors (TCRs). Interactions provided by accessory receptors, such as signaling lymphocytic activation molecule (SLAM) and Ly108, on selecting DP cells provide key signals mediated by SAP, Fyn and perhaps NF-kB, to allow transition of iNKT cell progenitors to CD4+CD8loCD24hiCD69hi ‘Stage 0’ cells. Following positive selection, progenitors rapidly proliferate and progress down a developmental pathway as shown. The ability to produce IL-4 (Stage 2) precedes that of IFN-g (Stage 3). The acquisition of NK receptor expression (for example NK1.1) and ability to produce IFN-g can occur outside the thymus, although a population of resident thymic iNKT cells with this mature phenotype also exists. Egr2 appears to influence iNKT cell development as cells transit from Stage 0 to Stage 1. The transcription factor PLZF is important for transition from Stage 1 to Stage 2, whereas the transcription factors T-bet and GATA3, and the availability of IL-15, are important during the later transition to mature iNKT cells.
cell selection. Early Egr2-deficient iNKT cell progenitors expressed high levels of CD24 and were reported to upregulate CD69, suggesting that at least some cells were capable of positive selection. However, fewer cells exhibited low levels of CD24 and more retained an immature CD44–NK1.1– phenotype, implicating a partial block at the transition from Stage 0 to Stage 1 of ontogeny (Figure 1). Although an increased proportion of thymic iNKT cells appeared to be dividing, Egr2–/– mice had reduced numbers of iNKT cells at all stages of maturation. One possible explanation for this finding is that a higher proportion of immature CD24– Egr2–/– iNKT cell underwent apoptosis, perhaps leaving fewer cells to continue down the maturation pathway. Immunology and Cell Biology
Collectively, these findings highlight several important findings. First, these are the first data to show that the calcineurin–NFAT signaling pathway is critical for iNKT cell development. Although it is not surprising that calcium-dependent events might be required for iNKT cell ontogeny, before the current report, this notion had never been formally tested. Second, the authors demonstrate that the NFAT target gene Egr2, not the related genes Egr1 and Egr3, is essential for development of iNKT cells, but not conventional T cells. Thus, Egr2 has nonoverlapping functions with other Egr family members during iNKT ontogeny. Third, the immature and apoptotic phenotype of Egr2–/– iNKT cells suggests that Egr2 is required early in devel-
opment to support the survival and perhaps maturation of iNKT cells at, or immediately following, positive selection. The current studies make possible several interesting avenues of investigation regarding the function of Egr2 in iNKT cell development and activation. Importantly, it is not clear from the data provided whether Egr2 function is required in iNKT cell progenitors, ‘selecting’ DP cells or both cell populations. This question could be addressed through generation and examination of bone marrow chimeric mice using congenically marked Egr2-deficient fetal liver cells in combination with WT hematopoietic precursors. Failure of WT ‘selecting’ cells to rescue iNKT cell development would confirm a cell-intrinsic function
News and Commentary 363
for Egr2 in iNKT progenitors. It is also important to determine whether Egr2–/– thymocytes express critical accessory receptors and signaling molecules required for early iNKT development. For example, the Src family tyrosine kinase Fyn and the adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) interact to promote signaling downstream of SLAM and Ly108 receptors during iNKT cell development. Defects in the SLAM receptor–SAP–Fyn signaling axis lead to profound defects in iNKT cell ontogeny, with cells blocked at a very early stage of development.11 Thus, it would be important to determine whether loss of Egr2 affects the expression or function of molecules in this signaling pathway, or putative downstream targets such as NF-kB, in developing iNKT cells progenitors and/or DP cells. Egr2-deficient thymocytes express normal levels of transcripts encoding specific survival factors (such as the cytokines IL-2, IL-7 and IL-15 and the prosurvival molecules Bcl-2 and BclXL) and apoptosis promoting molecules (FasL, Bim, Bak and Bax). Thus, one is left to ponder the cause of increased cell death in Egr2–/– iNKT cells. Examination of other specific candidates, or use of nonbiased approaches such as gene expression profiling or chromatin immunoprecipitation, may provide additional insights into the Egr2-
dependent mechanisms controlling iNKT cell survival. Interestingly, Egr2 is a negative regulator of T-cell activation12 and essential for induction of T-cell anergy.13,14 Therefore, it will also be informative to further explore Egr2’s function during mature iNKT cell activation. Although the authors demonstrate that the few iNKT cells remaining in Egr2–/– mice exhibit subtle defects in cytokine production, it is not clear whether these defects result from absent Egr2 expression or altered thymic ontogeny. Future efforts to silence the expression of Egr2 in mature iNKT cells will establish whether this transcription factor is required for iNKT cell homeostasis and to mediate specific functions, such as cytokine production, cytotoxicity and costimulation. These results of the current and future studies are likely to provide clues into how iNKT cell numbers or functions can be manipulated to enhance host immunity and provide protection against asthma, cancer and autoimmunity.
4 5
6 7
8
9
10
11
12
13 1
2 3
Tupin E, Kinjo Y, Kronenberg M. The unique role of natural killer T cells in the response to microorganisms. Nat Rev Microbiol 2007; 5: 405–417. Terabe M, Berzofsky JA. The role of NKT cells in tumor immunity. Adv Cancer Res 2008; 101: 277–348. Meyer EH, DeKruyff RH, Umetsu DT. iNKT cells in allergic disease. Curr Top Microbiol Immunol 2007; 314: 269–291.
14
Nowak M, Stein-Streilein J. Invariant NKT cells and tolerance. Int Rev Immunol 2007; 26: 95–119. Lazarevic V, Zullo AJ, Schweitzer MN, Staton TL, Gallo EM, Carbtree GR et al. 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. Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol 2007; 25: 297–336. Savage AK, Constantinides MG, Han J, Picard D, Martin E, Li B et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 2008; 29: 391–403. Macian F. NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol 2005; 5: 472–484. Bueno OF, Brandt EB, Rothenberg ME, Molkentin JD. Defective T cell development and function in calcineurin A beta-deficient mice. Proc Natl Acad Sci USA 2002; 99: 9398–9403. Neilson JR, Winslow MM, Hur EM, Crabtree GR. Calcineurin B1 is essential for positive but not negative selection during thymocyte development. Immunity 2004; 20: 255–266. Griewank K, Borowski C, Rietdijk S et al. Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development. Immunity 2007; 27: 751–762. Safford M, Collins S, Lutz MA, Allen A, Huang CT, Kowalski J et al. Egr-2 and Egr-3 are negative regulators of T cell activation. Nat Immunol 2005; 6: 472–480. Zhu B, Symonds AL, Martin JE, Kioussis D, Wraith DC, Li S et al. Early growth response gene 2 (Egr-2) controls the self-tolerance of T cells and prevents the development of lupuslike autoimmune disease. J Exp Med 2008; 205: 2295–2307. Harris JE, Bishop KD, Phillips NE, Mordes JP, Greiner DL, Rossini AA et al. Early growth response gene-2, a zinc-finger transcription factor, is required for full induction of clonal anergy in CD4+ T cells. J Immunol 2004; 173: 7331–7338.
Immunology and Cell Biology
