Id3 Restricts δγ NKT Cell Expansion by Controlling Egr2 and c-Myc Activity Baojun Zhang, Anjun Jiao, Meifang Dai, David L. Wiest and Yuan Zhuang This information is current as of September 30, 2021. J Immunol published online 16 July 2018 http://www.jimmunol.org/content/early/2018/07/15/jimmun ol.1800106 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/07/15/jimmunol.180010 Material 6.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 30, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 16, 2018, doi:10.4049/jimmunol.1800106 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 transcription factor 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-protein activity in mice. However, how E proteins 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 genes 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 Downloaded from blocked PLZF upregulation. We further show that this Egr2-mediated gd NKT cell expansion is dependent on c-Myc. c-Myc 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: 000–000. http://www.jimmunol.org/ he gd T cells are generated from fetal development through as in immune disorders such as Sjo¨gren’s syndrome (14), dermatitis 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 September 30, 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 MembersoftheEgrfamily(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 2 Id3 CONTROLS gd NKT CELL EXPANSION 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.