Id2 Collaborates with Id3 To Suppress Invariant NKT and Innate-like Tumors Jia Li, Sumedha Roy, Young-Mi Kim, Shibo Li, Baojun Zhang, Cassandra Love, Anupama Reddy, Deepthi This information is current as Rajagopalan, Sandeep Dave, Anna Mae Diehl and Yuan of October 3, 2021. Zhuang J Immunol 2017; 198:3136-3148; Prepublished online 3 March 2017; doi: 10.4049/jimmunol.1601935 http://www.jimmunol.org/content/198/8/3136 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/03/03/jimmunol.160193 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 95 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/198/8/3136.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 3, 2021 • No Triage! 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The Journal of Immunology Id2 Collaborates with Id3 To Suppress Invariant NKT and Innate-like Tumors Jia Li,*,1 Sumedha Roy,*,1 Young-Mi Kim,† Shibo Li,† Baojun Zhang,* Cassandra Love,‡ Anupama Reddy,‡ Deepthi Rajagopalan,‡ Sandeep Dave,‡ Anna Mae Diehl,x and Yuan Zhuang* Inhibitor of DNA binding (Id) proteins, including Id1–4, are transcriptional regulators involved in promoting cell proliferation and survival in various cell types. Although upregulation of Id proteins is associated with a broad spectrum of tumors, recent studies have identified that Id3 plays a tumor-suppressor role in the development of Burkitt’s lymphoma in humans and hepatosplenic T cell lymphomas in mice. In this article, we report rapid lymphoma development in Id2/Id3 double-knockout mice that is caused by unchecked expansion of invariant NKT (iNKT) cells or a unique subset of innate-like CD1d-independent T cells. These populations began to expand in neonatal mice and, upon malignant transformation, resulted in mortality between 3 and 11 mo Downloaded from of age. The malignant cells also gave rise to lymphomas upon transfer to Rag-deficient and wild-type hosts, reaffirming their inherent tumorigenic potential. Microarray analysis revealed a significantly modified program in these neonatal iNKT cells that ultimately led to their malignant transformation. The lymphoma cells demonstrated chromosome instability along with upreg- ulation of several signaling pathways, including the cytokine–cytokine receptor interaction pathway, which can promote their expansion and migration. Dysregulation of genes with reported driver mutations and the NF-kB pathway were found to be shared between Id2/Id3 double-knockout lymphomas and human NKT tumors. Our work identifies a distinct premalignant state and http://www.jimmunol.org/ multiple tumorigenic pathways caused by loss of function of Id2 and Id3. Thus, conditional deletion of Id2 and Id3 in developing T cells establishes a unique animal model for iNKT and relevant innate-like lymphomas. The Journal of Immunology, 2017, 198: 3136–3148. significant portion of cancer research is dedicated to the and sensitivity to therapy (7). Therefore, they are deemed as at- identification of underlying factors that contribute to the tractive tumor therapeutic targets based on the use of small mol- A hallmarks of tumorigenesis (1–3), such as dysregulated ecule inhibitors and Id-binding peptides in mouse models and proliferation and self-renewal (4, 5). All four members of the cancer cell lines (10, 13–16). In contrast, deep sequencing of inhibitor of DNA binding (Id) family (Id1–4) of helix–loop–helix human Burkitt’s lymphoma samples has revealed loss-of-function by guest on October 3, 2021 transcription factors share the ability to promote proliferation and mutations in Id3 in a large subset of patients, supporting the a stem cell–like dedifferentiated state (6, 7) and are often upreg- tumor-suppressor role of Id3 in some contexts (17–19). Id32/2 ulated in various cancer types (8–12). Id protein activity is also mice have also been reported to develop gd hepatosplenic T cell found to be directly correlated with tumor initiation, progression, lymphoma as a consequence of Vg1.1+Vd6.3+ gd T cell pop- ulation expansion (20). Although there are some reports sug- gesting a context-dependent role for Id4 in tumor progression or *Department of Immunology, Duke University Medical Center, Durham, NC 27710; suppression (21–23), there is only limited evidence in favor of a †Department of Pediatrics, Oklahoma University Health Sciences Center, Oklahoma City, OK 73014; ‡Duke Institute for Genome Sciences and Policy, Duke University, tumor-suppressive role for Id2 (24). Durham, NC 27710; and xDepartment of Medicine, Duke University Medical Center, Id proteins are primarily considered inhibitors of E proteins, the Durham, NC 27710 founding members of basic helix–loop–helix transcription factors 1 J.L. and S.R. contributed equally to this work. (25). Id2 and Id3, which are highly expressed in lymphocytes, are ORCIDs: 0000-0003-3070-8635 (J.L.); 0000-0002-2766-0071 (Y.-M.K.); 0000- known for their critical roles in suppressing E protein activity at 0002-7786-4304 (B.Z.). various stages to allow conventional ab T cell development (26, Received for publication November 14, 2016. Accepted for publication February 7, 2017. 27). They have also been recently described to repress innate-like gd and invariant NKT (iNKT) cell development (28–37). This work was supported by National Institutes of Health Grants R01 GM059638 and P01 AI102853 (to Y.Z.). Innate-like T cells are unique populations of T cells that derive The microarray data presented in this article have been submitted to the Gene Ex- their name from an innate cell–like ability to quickly secrete a pression Omnibus repository (https://www.ncbi.nlm.nih.gov/projects/geo) under ac- myriad of cytokines in response to Ag (38, 39). These populations cession number GSE83761. play key roles in providing protection against tumors and certain Address correspondence and reprint requests to Prof. Yuan Zhuang, Jones Building, infectious and autoimmune diseases, even though they are usually Room 326, 207 Research Drive, Duke University, Durham, NC 27710. E-mail ad- dress: [email protected] present in negligible proportions compared with conventional The online version of this article contains supplemental material. T cells (40, 41). The best characterized innate-like T cells are Abbreviations used in this article: CD1dTet, CD1d tetramer; DN, double-negative; Vg1.1Vd6.3 gd T cells and iNKT cells, but other cell types, like DP, double-positive; Id, inhibitor of DNA binding; iNKT, invariant NKT; L-DKO, mucosal-associated invariant T cells, are also included in this f/f f/f + Id2 Id3 LckCre ; PLZF, promyelocytic leukemia zinc finger; SOM, self-organizing category (42). iNKT cells express a semi-invariant TCR, Va14Ja18, map; SP, single-positive; TKO, Id2f/fId3f/fLckCre+CD1d2/2; WT, wild-type. which allows them to be identified by a-GalCer–loaded CD1d Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 tetramers (CD1dTets) (43). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601935 The Journal of Immunology 3137 In this article, we describe a rapid generation of iNKTor innate-like Bioinformatics and statistical analysis lymphoid tumors upon deletion of Id2 and Id3 in thymocytes. We Microarray data for premalignant and lymphoma cells were normalized also delineate the expanding precursor populations and dysregulated using RMA, and differential analysis was done using the limma package pathways that account for lymphoma development. Given that NKT available through Bioconductor (45). Publicly available normalized lymphomas are extremely rare and highly lethal in humans (44), our ImmGen data for WT cells were requested and downloaded from http:// study provides a much needed animal model for understanding the rstats.immgen.org/DataRequest/ (46). The two normalized datasets were combined according to the Empirical Bayes method using the Web tool genetic basis of similar types of tumors in humans. ArrayMining (http://www.arraymining.net) (47). Materials and Methods Data plotting, visualization, and statistics Mice Gene-expression (fold change) heat maps were generated using Gene-E (http://www.broadinstitute.org/cancer/software/GENE-E/). Self-organizing f/f f/f + Id2 Id3 LckCre (L-DKO) mice were generated as previously described maps (SOM) were generated by the Partek Genomics Suite made available 2/2 (34). CD1d mice were purchased from the Jackson Laboratory (strain by the Duke Center for Genome and Computational Biology. Principal f/f f/f + 008881) and were bred with L-DKO mice to generate Id2 Id3 LckCre component analysis was performed and plotted using the built-in R func- 2/2 CD1d (TKO) mice. All mice were bred in a specific pathogen–free tions prcomp and plot3d in open-source RStudio software. Gene overlaps facility at the Duke