Id3 Restricts δγ NKT Cell Expansion by Controlling Egr2 and c- Activity Baojun Zhang, Anjun Jiao, Meifang Dai, David L. Wiest and Yuan Zhuang This information is current as of October 2, 2021. J Immunol 2018; 201:1452-1459; Prepublished online 16 July 2018; doi: 10.4049/jimmunol.1800106 http://www.jimmunol.org/content/201/5/1452 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Id3 Restricts gd NKT Cell Expansion by Controlling Egr2 and c-Myc Activity

Baojun Zhang,*,† Anjun Jiao,* Meifang Dai,† David L. Wiest,‡ and Yuan Zhuang† gd NKT cells are neonatal-derived gd T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the PLZF (promyelocytic leukemia zinc finger). Previ- ous studies have demonstrated that the population size of gd NKT cells is tightly controlled by Id3-mediated inhibition of E- activity in mice. However, how E promote gd NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating gd NKT cell expansion. We observed a higher expression of Egr family in gd NKT cells compared with the conventional gd T cell population. Loss of function of Id3 caused an expansion of gd NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient gd NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated gd NKT cell expansion is dependent on c-Myc. c-Myc Downloaded from knockdown attenuated the proliferation of Id3-deficient gd NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2–double-deficient gd NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2–Id3–E2A, which normally restricts the population size of gd NKT cells by adjusting Egr2 dosage and c-Myc expression. The Journal of Immu- nology, 2018, 201: 1452–1459.

he gd T cells are generated from fetal development through as in immune disorders such as Sjo¨gren’s syndrome (14), dermatitis http://www.jimmunol.org/ adult life in a series of developmental waves (1, 2). Each (8), and asthma (15, 20). T wave of gd T cells preferentially migrates to particular The population size of gd NKT cells is tightly regulated during anatomical locations and serves site-specific immune regulatory thymic development and typically represents 10% of total gd T cells functions ranging from tissue homeostasis to immunosurveillance in C57BL/6 mice. gd NKT cells undergo a dramatic expansion in (1, 3–5). The Vg1.1Vd6.3 gd T cells represent the late fetal wave that Id32/2 mice, particularly on the background where Id2 expression is expands during the neonatal window. These gd T cells were first also partially compromised (11, 21–24). This phenomenon can be described by Pereira and colleagues (6) in 1997 and later referred to explained by elevated activity of E proteins because deletion of E2A as NKTgd T cells (7, 8) or gd NKT cells (9, 10). This population in Id3-deficient mice attenuates gd NKT cell expansion (21, 23, 24). by guest on October 2, 2021 develops in the neonatal thymus and migrates to peripheral sites such Furthermore, a number of laboratories have observed an enhanced as the spleen, lymph nodes, and, most prominently, the liver (6). gd development of gd NKT cells in mouse strains with deficiencies or NKT cells are considered an innate-like lineage based on expression impairments in TCR downstream molecules, such as SLP-76 (11, 25) of the innate signature transcription factor PLZF (8, 11). Mature gd and IL-2–inducible T cell kinase (10, 15). Dok1, as an inhibitor of NKT cells are phenotypically CD24lowCD44highNK1.1+ and are ca- ZAP-70, LAT, SLP-76, Akt, and Erk1/2 (26–29), consistently pro- pable of secreting various cytokines of IFN-g, IL-4, and IL-13 upon motes gd NKT cell development and expansion (28). Because Id3 is TCR stimulation (8, 12–16). These innate features endow gd NKT the most downstream factor in the TCR signaling pathway, it is cells with important functions in immune protection (17–19) as well possible that defects in these upstream signaling molecules may prevent proper activation of Id3, which consequently results in elevated E-protein activity. Therefore, a common mechanism un- *Department of Pathogenic Microbiology and Immunology, School of Basic Medical derpinning the gd NKT expansion reported in these studies may be Sciences, Xi’an Jiaotong University, Xi’an, ShaanXi 710061, China; †Department E-protein mediated. Such a regulatory mechanism of gd NKT de- of Immunology, Duke University Medical Center, Durham, NC 27710; and velopment and expansion has not yet been explored. ‡Blood Cell Development and Function Program, Fox Chase Cancer Center, Phila- delphia, PA 19111 Members of the Egr family (Egr1, Egr2,andEgr3)senseTCR ORCIDs: 0000-0002-7786-4304 (B.Z.); 0000-0002-5688-818X (A.J.); 0000-0002- signaling through p-ERK and transmit TCR signals through the 0792-3188 (D.L.W.); 0000-0002-2964-3654 (Y.Z.). upregulation of Id3, which in turn inhibits E-protein activity. TCR Received for publication January 24, 2018. Accepted for publication June 21, 2018. signal strength and duration of Egr activity are considered major This work was supported by National Institutes of Health Grants R01GM-059638 (to Y.Z.) factors distinguishing ab from gd T lineage at the preTCR check- and P01AI102853 (to D.L.W. and Y.Z.), National Basic Research Program of China Grant point, with a weaker signal instructing ab T lineage and a stronger 81771673 (to B.Z.), and the Thousand Young Talents Program of China (to B.Z.). signal supporting gd T lineage development (30). Egr family mem- Address correspondence and reprint requests to Dr. Baojun Zhang or Dr. Yuan bers can also regulate the proliferation and survival of lymphocytes Zhuang, Xi’an Jiaotong University, 76 Yanta West Road, Xi’an, Shaanxi 710061, China (B.Z.) or Department of Immunology, Duke University Medical Center, 328 (31–33). A recent study has also shown that sustained induction of Jones Building, 207 Research Drive, Box 3010, Durham, NC 27710 (Y.Z.). E-mail Egr2 in response to TCR signaling in invariant NKT (iNKT) cells addresses: [email protected] (B.Z.) and [email protected] (Y.Z.) results in upregulation of PLZF, which is crucial for an innate de- The online version of this article contains supplemental material. velopmental program (34). Whether the same mechanism is involved Abbreviations used in this article: iNKT, invariant NKT; shRNA, short hairpin RNA; in gd NKT cell development remains to be determined. Furthermore, WT, wild type. the relationship between the Egr2–PLZF pathway and E2A-mediated Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 gd NKT cell development and expansion has not been evaluated. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800106 The Journal of Immunology 1453

In this study we have examined the role of Egr proteins, namely (5 ng/ml IL-7, 1 ng/ml IL-2, and 5 ng/ml IL-15). Id3-deficient or Id3/Egr2– Egr1, Egr2, and Egr3, in gd NKT cell development and found that double-deficient gd T cells were infected with c-Myc short hairpin RNA Egr2 plays a prominent role in gd NKT cell expansion. We (shRNA) and c-Myc overexpression virus, respectively. Forty-eight hours later, cells were collected for RNA or BrdU analysis. The shRNA demonstrate that Id3 deletion results in upregulation of Egr family (59-TGTAAGCTTCAGCCATAATTT-39) was designed to target the 39UTR of genes and PLZF among the expanded gd NKT cells. Deletion of c-Myc and was cloned into a vector carrying a miR30-based backbone (38). Egr2 ameliorated the expansion of Id3-deficient gd NKT cells and Statistical analysis prevented PLZF upregulation. We further show that Egr2 pro- motes gd NKT cell proliferation through c-Myc. Collectively, we The data were compared using Student t test, with p , 0.05 considered , , revealed a regulation loop of Egr2–Id3–E2A in regulating gd NKT significant and p 0.01 and p 0.001 considered highly significant. cell development and expansion. Results 2/2 Materials and Methods Egr2 controls the expansion of Id3 gd NKT cells Mice and reagents Previous studies have shown that Id3 deficiency causes a dramatic Mice with Id3 conditional knockout (35) and LckCre transgenic (36) alleles expansion of gd NKT cells over conventional gd T cells in the have been described previously. Egr12/2,Egr2f/f, and Egr32/2 mice were as neonatal thymus. We hypothesized that the loss of Id3 function previously reported (37). All the strains are on B6 background. Animals were leads to the activation of an innate-associated transcription program. bred and maintained in the SPF facility managed by the Duke University We therefore decided to search for transcription factors with critical Division of Laboratory Animal Research. Animal procedures were approved roles in regulating innate-like T cell development. Egr2 has been by the Duke University Institutional Animal Care and Use Committee. shown to support gd lineage differentiation by mediating the TCR The Abs used were as follows: FITC anti-mouse TCRd (GL3), PE/Cy7 anti- Downloaded from mouse TCRd (GL3), PE/Cy5 anti-mouse NK1.1 (PK136), PE/Cy7 anti-mouse/ signal (34, 37, 39). We noted that Egr2 expression was also higher human CD44 (IM7), APC anti-mouse CD24 (M1/69), PE-Cy7 anti-mouse IFN-g in gd NKT cells than in any other subtypes of gd Tcells,as (XMG1.2), and Pacific Blue anti-mouse IL-17 (TC11-18H10.1) (all purchased reported in The Immunological Genome Project. This prompted us from BioLegend); PE anti-mouse Vd6.3/2 (8F4H7B7), APC anti-mouse PLZF (9E12), and APC BrdU Flow Kit (all purchased from BD Biosciences); and to test the possibility that Egr2 may be involved in differentially rabbit anti-mouse Egr2 (EPR4004) and goat anti-rabbit IgG H&L (Alexa Fluor regulating gd NKT cells versus other gd T cells. 488, ab150077) (both purchased from abcam). To test whether Egr2 plays a necessary role in supporting gd NKT cell expansion in Id3-deficient mice, we first verified the reported http://www.jimmunol.org/ Flow cytometry Egr2 expression patterns in gd NKT cells (Vg1.1+Vd6.3+)and For cell surface analysis, single cells from the thymus were suspended in conventional gd T cells (Vg1.1+Vd6.32) purified from thymi of wild 3 cold FACS buffer (1 PBS supplemented with 5% bovine calf serum). A type (WT) neonatal mice. Indeed, Egr2 expression was higher among total of 5 3 106 cells were stained with Abs in the dark at 4˚C for 30 min. After washing with cold FACS buffer, cells were analyzed using a gd NKT cells than in conventional gd T cells (Fig. 1A–C). Egr3, but FACSCanto II flow cytometer (BD Biosciences). FlowJo software (Tree not Egr1, also displayed a relatively higher level of transcripts in gd Star) was used for data analysis. In some experiments, gd T cells were NKT cells than in the control gd population. Within the gd NKT cell sorted with a MoFlo XDP cell sorter. lineage, Id3 deletion resulted in upregulation of all three Egr genes at For cytokine analysis, thymocytes were stimulated in vitro with PMA/

mRNA levels (Fig. 1A–C). We further confirmed an upregulation of by guest on October 2, 2021 Ionomycin in the presence of brefeldin A and monensin for 4 h. Cells were washed and stained with anti-TCRd and Vd6.3/2 Abs. After a 30-min Egr2 protein level specifically among Id3-deficient gd NKT cells incubation, cells were fixed and permeabilized according to BD Biosci- (Supplemental Fig. 1). We then tested if any of the Egr genes are ences Cytofix/Cytoperm Fixation/Permeabilization Kit protocol, followed required for gd NKT cell development and/or expansion in Id3- by IFN-g and IL-17 analysis with FACS. deficient mice. Deletion of Egr2,butnotEgr1 and Egr3,signifi- Real-time PCR analysis cantly reduced numbers of gd NKT cells in Id3-deficient mice. De- letion of both Egr2 and Egr3 reduced the population size of gd NKT 2 + Total RNA was extracted from purified Vd6.3 and Vd6.3 gd T cells with cells in Id3-deficient mice to WT levels, indicating that Egr2 and RNAqueous micro kit (Life Technologies). Reverse transcription was performed with Moloney murine leukemia virus reverse transcriptase (Life Egr3 work together to promote gd NKT cell expansion in response to Technologies). SYBR-based real-time PCR was performed to quantitatively elevated E-protein activity (Fig. 1D, 1E). Moreover, because Egr2/3 compare expression, with results normalized by b-actin expression. deficiency returns the number of gd NKT cells generated in Id3/Egr2/ Quantitative PCR primer sequences are as follows: Egr1 forward primer: Egr3 triple knockout mice to WT levels without completely elimi- 59-AGCGCCTTCAATCCTCAAG-39; Egr1 reverse primer: 59-TTTGGCTG- nating them, Egr2 and Egr3 do not appear to be required for the GGATAACTCGTC-39; Egr2 forward primer: 59-TTGACCAGATGAAC- GGAGTG-39; Egr2 reverse primer: 59-TGCCCATGTAAGTGAAGGTC-39; generation of gd NKT cells. We then further tested the involvement Egr3 forward primer: 59-TGCCCCAACCGCCGCTTACTCTCA-39; Egr3 of Egr2 in gd NKT cell proliferation using in vivo BrdU pulse la- forward primer: 59-GGCGCACCCCCTTTCTCCGACTTC-39; PLZF for- beling. We have shown in previous publications that Id3 deficiency ward primer: 59-CCACCTTCGCTCACATACAG-39; PLZF reverse primer: resultsinvigorousproliferation of neonatal gd NKT cells (22). Egr2/ 59-CACAGCCATTACACTCATAGGG-39; c-Myc forward primer: 59-GCT- GTTTGAAGGCTGGATTTC-39; and c-Myc reverse primer: 59-GATGA- Id3 double deficiency reduced the proliferation rate of gd NKT cells, AATAGGGCTGTACGGAG-39. relative to that in Id3-deficient mice (Fig. 1F–H). These findings demonstrate that Egr2 expression is essential for regulating the ex- BrdU incorporation pansion of gd NKT cells in Id3-deficient mice. Additionally, Egr3 For in vivo experiments, 50 ml (100 mg) BrdU was injected i.p. to neonatal mice collaborates with Egr2 in promoting gd NKT cell expansion. for 4 h of pulse labeling. For in vitro experiments, cultured gd T cells were incubated with BrdU at a final concentration of 10 mM for 30 min. Single cells Egr2 significantly affected maturation but not effector from thymus (in vivo) or culture (in vitro) were stained with anti-TCRd,Vd6.3/2, characteristics of Id3-deficient gd NKT cells and BrdU Abs, according to the manufacturer’s instructions (BD Biosciences). The stained cells were analyzed on a FACSCanto II flow cytometer. Maturation and effector differentiation are two major steps in gd T cell development. CD24 downregulation has been well defined as Retrovirus transduction a critical step in T cell maturation. We found that the majority of gd Vd6.3+ gd T cells from the thymi of Id3-deficient or Id3/Egr2–double-deficient NKT cells in Id3-deficient mice are CD24 negative (Fig. 2A). This neonatal mice were sorted by FACS and cultured overnight with 5 mg/ml phenotype remains unchanged upon further deletion of Egr2 on an anti-CD3 and anti-CD28 Abs in the presence of a mixture of cytokines Id3-deficient background. Once gd NKT cells become matured, they 1454 Id3 CONTROLS gd NKT CELL EXPANSION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 1. Egr2 is essential to gd NKT cell development in Id32/2 mice. (A–C) Transcription analysis of Egr1, Egr2, and Egr3 in conventional and gd NKT cells from indicated strains. (D) Percentage of gd NKT cells in the thymus of indicated mouse strains. (E) Statistical summary for (D). (F) Absolute cell number of gd NKT cells in the thymus of indicated mouse strains. (G) Representative FACS plots for BrdU incorporation of thymic gd NKT cells from mouse strains. (H) Summary for the percentage of BrdU+ gd NKT cells in the thymus from mouse strains. The data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. typically enter effector phase, expressing surface markers such as percentage, but not absolute numbers, of CD44+NK1.12 and CD44+ CD44 and NK1.1 and secreting cytokines such as IFN-g (10, 40). We NK1.1+ gd NKT cells (Fig. 2B–D). Furthermore, we found that loss of found a similar pattern of CD44 and NK1.1 expression between WT Id3 increased the frequency of IL-17 producers but not IFN-g pro- and Id32/2 gd NKT cells (Fig. 2B). Deletion of Egr2 augmented the ducers among total gd NKT cells (Fig. 2E, 2F). This effector pattern The Journal of Immunology 1455 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Effect of Egr2 on the maturation and effector feature of Id32/2 gd NKT cells. (A) CD24 expression in gd NKT cells from the indicated mouse strains. (B) CD44 and NK1.1 expression in gd NKT cells from the indicated mouse strains. (C) Statistical summary for (A)and(B). (D) The absolute number of gd NKT cells in indicated stages. (E) Representative FACS plots for IFN-g and IL-17 expression in gd NKT cells of thymus. (F) Summary for the percentage of IFN-g+ and IL-17+ cells in thymic gd NKT cells. The data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. was not affected by further deletion of Egr2. Our data demonstrated (34). To address whether Egr2 can control PLZF expression in Id32/2 that functions of Egr2 in gd NKT cells promote the maturation without cells, we compared RNA and protein levels of PLZF in Id32/2 and dramatically influencing the effector features of these cells. Id32/2Egr22/2 gd NKT cells. We found a 3-fold increase of PLZF transcription in Id32/2 gd NKT cells compared with WT gd NKT Egr2 is required for increased PLZF expression in 2/2 2/2 cells (Fig. 3A). Egr2 deficiency returned PLZF transcription in Id3 Id3 gd T cells gd NKT cells to WT levels. Likewise, intracellular staining for PLZF PLZF is a key transcription factor that controls the differentiation of protein revealed that Id3 deficiency markedly increased the PLZF multiple innate populations, including gd NKT and iNKT cells (11, 41, protein levels in gd NKT cells compared with WT gd NKT cells, and 42). PLZF has been shown to be a direct target of Egr1/2 in iNKT cells this was reversed by Egr2 deficiency (Fig. 3B). This suggested a 1456 Id3 CONTROLS gd NKT CELL EXPANSION

FIGURE 3. PLZF expression controlled by Egr2 in Id32/2 gd NKT cells. (A) The tran- scription of PLZF in thymic gd NKT cells from indicated mouse strains. (B) FACS analysis of PLZF protein expression in thymic gd NKT cells from indicated mouse strains. The data are representative of three independent mice. *p , 0.05, **p , 0.01, ***p , 0.001.

common role for Egr2 in regulating expression levels of PLZF in both In this study, we explored whether c-Myc is involved in the iNKT cells and gd NKT cells. So far, our data demonstrated that in gd Egr2-driven Id32/2 gd NKT cell proliferation. We found that NKT cells, Egr2 lies upstream of PLZF but is also controlled by a Id32/2 gd NKT cells exhibited a significant increase in c-Myc feedback regulation of the Id3/E2A axis. transcription compared with WT cells (Fig. 4A). Egr2 deficiency mildly, but significantly, decreased c-Myc transcription. How- c-Myc is responsive to the Egr2–Id3–E2A axis and is required Downloaded from 2/2 ever, knockdown of c-Myc by shRNA did not affect Egr2 tran- for Id3 gd NKT cell proliferation scription (Fig. 4B, 4C). These data demonstrate that loss of Id3 c-Myc plays a critical role in cell cycle regulation (43) and has enhances c-Myc transcription partially through the upregulation been reported to be an E2A target in a lymphoma cell line (44). of Egr2. However, elevation of E-protein activity in Id3-deficient http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. c-Myc is responsible to Egr2 and required for gd NKT cell proliferation. (A) The transcription of c-Myc in gd NKT cells of indicated mouse strains. (B) Knockdown of c- Myc with retrovirus-based shRNA. (C) Effect of c-Myc knockdown on Egr2 transcription. (D)HistogramofBrdU incorporation in Id32/2 gd NKT cells transduced with mock or shRNA retrovirus. (E) Statistical summary for (D). (F) Histogram of BrdU incorporation in Id32/2Egr22/2 gd NKT cells transduced with mock or c-Myc overexpressing retrovirus. (G) Statistical summary for (F). The data are representative of three inde- pendent experiments. **p , 0.01, ***p , 0.001. The Journal of Immunology 1457

FIGURE 5. The feedback regulation loop of Egr2–Id3–E2A controls the development and expansion of gd NKT cells. Sensing proximal TCR signaling, a cassette of downstream signaling is activated, including p-Erk, Egr, Id3, and E2A molecules. The dosage of E proteins is critical for gd T cell lineage development. Left, Normally, p-Erk can activate Egr genes, including Egr1, Egr2, and Egr3, then induce Id3 expression to suppress E-protein activity. The low dose of E proteins could induce medium levels of Egr and PLZF and lead to the development of conventional gd T cells and a very

small population (1/10 in total gd T cells) of gd NKT cells. Right, When Id3 is missing, the inhibition of E-protein activity is released, which strongly Downloaded from induces Egr2, Egr3, and PLZF expression to enhance gd NKT cell differentiation. Meanwhile, strong E-protein activity and Egr2 expression could elevate c-Myc expression to cause a dramatic expansion of gd NKT cells. Therefore, the regulation loop of Egr2–Id3–E2A controls gd NKT cell lineage differentiation and population size. gd NKT cells also appears to contribute to c-Myc induction in the expansion phenotype of gd NKT cells in Id32/2 mice (24). ways that are independent of Egr2. Furthermore, by analyzing TCR rearrangements in Id32/2 gd http://www.jimmunol.org/ To determine whether c-Myc contributes to Id32/2 gd NKT NKT cells, Verykokakis et al. (24) showed that the junction cell proliferation, we purified gd NKT cells from the thymus of sequences of Vg1.1–Jg4 and Vd6–Jd1 segments were highly neonatal Id32/2 mice, stimulated them with anti-CD3 and anti- narrowed and conserved. Additionally, the rearrangements exhibit CD28 Abs in the presence of cytokine mixtures, and then the neonatal characteristic of lacking N nucleotide additions. transduced them with either mock or c-Myc shRNA for 48 h. We Collectively, these evidences demonstrate that this population found that, compared with the mock control, knockdown of entered a robust proliferation primarily at the neonatal, but not c-Myc significantly inhibited BrdU incorporation (Fig. 4D, 4E). the adult, stage. The basis for this stage-restricted proliferation Conversely, overexpression of c-Myc in Id32/2Egr22/2 gd NKT phenotype remains unclear. by guest on October 2, 2021 cells significantly enhanced proliferation, as evidenced by in- TCR signaling strength has been shown to correlate with creased BrdU incorporation (Figs. 4F, 4G). Therefore, c-Myc is lineage commitment to the ab or gd T cell fate, such that indeed involved in the proliferation of Id32/2 gd NKT cells and is strong TCR signaling favors gd T cell lineage differentiation controlled by high levels of E proteins and partially through Egr2 (37, 45–47). There is also evidence in support of strong TCR (Fig. 5). signaling promoting the innate lineage differentiation of gdNKT. However, there are quite a few controversial studies Discussion supporting that dysfunction in TCR downstream molecules, gd NKT cells, which exclusively express a Vg1.1-Vd6.3 TCR, such as Id3, KLF1, SLP-76, and inducible T cell kinase (10, 15), have been considered innate-like gd lineage, distinct from instead enhances expansion of gd NKT cells (11, 21–25). At the conventional gd T cell compartment, because of their least in Id32/2 mice, gd NKT cell expansion appears to be uniformly high expression of PLZF and effector molecules. The supported by a feedback loop comprising increased Egr2 and mechanisms that control gd NKT cell population size are still PLZF expression, which is consistent with the strong TCR unclear. Our study suggests that an Egr2–Id3–E2A feedback loop signaling–induced elevation and sustainability of Egr1/Egr2- controls gd NKT cell lineage expansion. Normally, gd TCR sig- enhanced NKT cell lineage development (34). It seems that naling activates a cascade that leads to phosphorylation of ERK the dysfunction in TCR downstream molecules could reinforce and Egr1/2/3 induction, which then represses E-protein activity the innate developmental pathway and allow an increase of in- through Id3, ultimately driving conventional gd lineage develop- nate lineage proliferation. gd NKT cells express a high level of ment. However, in this article, we report that elevated E-protein CD5 (8), an activation marker that is induced in proportion with activity in the absence of Id3 leads to upregulation of Egr2, PLZF, TCR signal strength. This idea is in line with the view that gd and c-Myc through a positive feedback loop, which then prefer- NKT cells are autoreactive and contribute to the occurrence of entially supports gd NKT lineage expansion. autoimmune diseases such as Sjo¨gren’s syndrome (14) and Id3 deficiency not only increased the proportion but also the would therefore normally be eliminated by negative selection absolute number of gd NKT cells. This suggests that a significant because of strong TCR signaling. Enforced expression of proliferation of gd NKT cells occurs in Id32/2 mice, which is also Vg1.1/Vd6.3 TCR has also been shown to be sufficient to found in the other mutant strains mentioned above. Indeed, BrdU support the generation of PLZF+ gd NKT cells, indicating that incorporation experiments showed that Id32/2 gd NKT cells from TCR signaling indeed plays a selective role in gd NKT lineage neonatal, not young adult, mice had a significantly increased differentiation (8). Given the complex relationship between the percentage of BrdU+ cells compared with WT (22). Consistent Vg1.1/Vd6.3 TCR and its downstream signaling events, further with this observation, adoptive transfer experiments employing investigation is needed to determine the signaling strength and adult bone marrow from WT and Id32/2 mice failed to replicate quality generated by the stereotypic Vg1.1/Vd6.3 TCR. 1458 Id3 CONTROLS gd NKT CELL EXPANSION

2 2 The expansion of gd NKT cells in Id3 / mice relies on high 12. Grigoriadou, K., L. Boucontet, and P. Pereira. 2003. Most IL-4-producing gamma delta thymocytes of adult mice originate from fetal precursors. levels of E proteins because of a release of Id3 inhibition (21, 23). J. Immunol. 171: 2413–2420. Although we found that Egr2 is responsive to high levels of 13. Alonzo, E. S., and D. B. Sant’Angelo. 2011. Development of PLZF-expressing E proteins and is required for gd NKT cell expansion, a luciferase innate T cells. Curr. Opin. Immunol. 23: 220–227. 14. Belle, I., J. Mahlios, A. McKenzie, and Y. Zhuang. 2014. Aberrant production of assay did not show evidence of E2A directly binding to the IL-13 by T cells promotes exocrinopathy in Id3 knockout mice. Cytokine 69: promoter region controlling Egr2 expression (data not shown). 226–233. c-Myc, as a cell cycle regulator in multiple cell types (43, 48), has 15. Felices, M., C. C. Yin, Y. Kosaka, J. Kang, and L. J. Berg. 2009. Tec kinase Itk in gammadeltaT cells is pivotal for controlling IgE production in vivo. Proc. Natl. been demonstrated to be one of E2A’s targets in a lymphoma cell Acad. Sci. USA 106: 8308–8313. line (44). We confirmed that c-Myc is responsive to high levels of E 16. Qi, Q., M. Xia, J. Hu, E. Hicks, A. Iyer, N. Xiong, and A. August. 2009. En- proteins, contributes to gd NKT cell proliferation, and is partially hanced development of CD4+ gammadelta T cells in the absence of Itk results in elevated IgE production. Blood 114: 564–571. dependent on Egr2 expression. Because Egr2 deficiency only par- 17. Belles, C., A. K. Kuhl, A. J. Donoghue, Y. Sano, R. L. O’Brien, W. Born, tially reduced c-Myc expression, and not to WT levels, it is likely that K. Bottomly, and S. R. Carding. 1996. Bias in the gamma delta T cell response to Listeria monocytogenes. V delta 6.3+ cells are a major component of the gamma the elevated levels of E-protein activity also regulate c-Myc in an delta T cell response to Listeria monocytogenes. J. Immunol. 156: 4280–4289. Egr2-independent manner. In addition to Myc, it is also of interest to 18. Matsuzaki, G., K. Hiromatsu, Y. Yoshikai, K. Muramori, and K. Nomoto. 1993. assess the relationship between the Id3/E2A axis and other pathways, Characterization of T-cell gamma delta T cells appearing at the early phase of murine Listeria monocytogenes infection. Immunology 78: 22–27. such as SAP and Lin28, which have also been shown to be required 19. Nakamura, T., G. Matsuzaki, and K. Nomoto. 1999. The protective role of T-cell for the development of gd NKT cells (11, 24, 49). However, these receptor Vgamma1+ T cells in primary infection with Listeria monocytogenes. signals, including TCR, are not sufficient for such a dramatic ex- Immunology 96: 29–34. pansion of gd NKT cells. We speculate that yet-to-be-identified 20. Mueller, C., and A. August. 2003. Attenuation of immunological symptoms of

allergic asthma in mice lacking the tyrosine kinase ITK. J. Immunol. 170: 5056– Downloaded from factors unique to the neonatal environment, such as specific Ags, 5063. nutrients, or signals, are essential to the expansion of gd NKT cells. 21. Ueda-Hayakawa, I., J. Mahlios, and Y. Zhuang. 2009. Id3 restricts the devel- opmental potential of gamma delta lineage during thymopoiesis. J. Immunol. Overall, our study revealed that the level of Egr2 expression 182: 5306–5316. controls the population size of gd NKT cells. Id3, as a down- 22. Zhang, B., M. Dai, Q. J. Li, and Y. Zhuang. 2013. Tracking proliferative history stream target of Egr2, is involved in a feedback regulation in lymphocyte development with cre-mediated sister chromatid recombination. PLoS Genet. 9: e1003887. to restrict the activity of Egr2 and thus to suppress the expansion 23. Zhang, B., Y. Y. Lin, M. Dai, and Y. Zhuang. 2014. Id3 and Id2 act as a dual of gd NKT cells. 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