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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 Rajagopalan, Sandeep Dave, Anna Mae Diehl and Yuan Zhuang
J Immunol April 15, 2017, 198 (8) 3136-3148; DOI: https://doi.org/10.4049/jimmunol.1601935
Jia Li
*Department of Immunology, Duke University Medical Center, Durham, NC 27710;
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Sumedha Roy
*Department of Immunology, Duke University Medical Center, Durham, NC 27710;
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Young-Mi Kim
†Department of Pediatrics, Oklahoma University Health Sciences Center, Oklahoma City, OK 73014;
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Shibo Li
†Department of Pediatrics, Oklahoma University Health Sciences Center, Oklahoma City, OK 73014;
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Baojun Zhang
*Department of Immunology, Duke University Medical Center, Durham, NC 27710;
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Cassandra Love
‡Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710; and
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Anupama Reddy
‡Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710; and
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Deepthi Rajagopalan
‡Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710; and
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Sandeep Dave
‡Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710; and
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Anna Mae Diehl
§Department of Medicine, Duke University Medical Center, Durham, NC 27710
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Yuan Zhuang
*Department of Immunology, Duke University Medical Center, Durham, NC 27710;
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Abstract

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 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 upregulation 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-κB pathway were found to be shared between Id2/Id3 double-knockout lymphomas and human NKT tumors. Our work identifies a distinct premalignant state and 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.

This article is featured in In This Issue, p.3003

Introduction

A significant portion of cancer research is dedicated to the identification of underlying factors that contribute to the hallmarks of tumorigenesis (1–3), such as dysregulated proliferation and self-renewal (4, 5). All four members of the inhibitor of DNA binding (Id) family (Id1–4) of helix–loop–helix transcription factors share the ability to promote proliferation and a stem cell–like dedifferentiated state (6, 7) and are often upregulated in various cancer types (8–12). Id protein activity is also found to be directly correlated with tumor initiation, progression, and sensitivity to therapy (7). Therefore, they are deemed as attractive tumor therapeutic targets based on the use of small molecule inhibitors and Id-binding peptides in mouse models and cancer cell lines (10, 13–16). In contrast, deep sequencing of human Burkitt’s lymphoma samples has revealed loss-of-function mutations in Id3 in a large subset of patients, supporting the tumor-suppressor role of Id3 in some contexts (17–19). Id3−/− mice have also been reported to develop γδ hepatosplenic T cell lymphoma as a consequence of Vγ1.1+Vδ6.3+ γδ T cell population expansion (20). Although there are some reports suggesting a context-dependent role for Id4 in tumor progression or suppression (21–23), there is only limited evidence in favor of a tumor-suppressive role for Id2 (24).

Id proteins are primarily considered inhibitors of E proteins, the founding members of basic helix–loop–helix transcription factors (25). Id2 and Id3, which are highly expressed in lymphocytes, are known for their critical roles in suppressing E protein activity at various stages to allow conventional αβ T cell development (26, 27). They have also been recently described to repress innate-like γδ and invariant NKT (iNKT) cell development (28–37).

Innate-like T cells are unique populations of T cells that derive their name from an innate cell–like ability to quickly secrete a myriad of cytokines in response to Ag (38, 39). These populations play key roles in providing protection against tumors and certain infectious and autoimmune diseases, even though they are usually present in negligible proportions compared with conventional T cells (40, 41). The best characterized innate-like T cells are Vγ1.1Vδ6.3 γδ T cells and iNKT cells, but other cell types, like mucosal-associated invariant T cells, are also included in this category (42). iNKT cells express a semi-invariant TCR, Vα14Jα18, which allows them to be identified by α-GalCer–loaded CD1d tetramers (CD1dTets) (43).

In this article, we describe a rapid generation of iNKT or innate-like lymphoid tumors upon deletion of Id2 and Id3 in thymocytes. We also delineate the expanding precursor populations and dysregulated pathways that account for lymphoma development. Given that NKT lymphomas are extremely rare and highly lethal in humans (44), our study provides a much needed animal model for understanding the genetic basis of similar types of tumors in humans.

Materials and Methods

Mice

Id2f/fId3f/fLckCre+ (L-DKO) mice were generated as previously described (34). CD1d−/− mice were purchased from the Jackson Laboratory (strain 008881) and were bred with L-DKO mice to generate Id2f/fId3f/fLckCre+CD1d−/− (TKO) mice. All mice were bred in a specific pathogen–free facility at the Duke University Division of Laboratory Animal Resources, and all procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee.

Flow cytometry analysis

Staining with surface marker Abs (BioLegend) was done before intracellular staining for promyelocytic leukemia zinc finger (PLZF) Ab (eBioscience) using a Foxp3 staining buffer set (eBioscience). CD1dTets were obtained from the Tetramer Facility of the National Institutes of Health. Flow cytometry analysis was performed on a FACSCanto II (BD Biosciences). Doublets and dead cells (7AAD+) were gated out before data analysis. Data were analyzed with FlowJo software (TreeStar).

Histopathological analysis

Tissue sections were removed immediately after sacrificing the mice and fixed in 10% PBS–formalin. Embedding, sectioning, and staining (H&E and Masson’s trichrome) were done by the Pathology Service Core at Duke University.

Adoptive transfer of lymphoma cells to Rag2−/− or wild-type hosts

Enlarged thymi from L-DKO donor mice were minced in PBS with 5% bovine calf serum, filtered, lysed for RBCs using BD Pharm Lyse lysing buffer (BD Biosciences), and washed with PBS. A total of 5 × 106 cells per recipient was injected into 6–8-wk-old Rag2−/− mice through their tail vein. Five- to seven-week-old wild-type (WT) mice were sublethally irradiated (300 rad) and injected with tumor cells 24 h later. Recipient mice were sacrificed 4–10 wk after transfer, and tissues were collected for FACS analysis and H&E staining.

PCR and real-time PCR

Genotyping of mice was done as described previously (34). Total RNA was extracted using an RNAqueous kit (Life Technologies), according to the manufacturer’s protocol, and reverse transcribed into cDNA using murine leukemia virus reverse transcriptase (Life Technologies). Real time PCR was performed using a FastStart DNA Master SYBR Green Kit, and quantitative expression was normalized to β-actin. The following forward and reverse genotyping primers were used and are listed in 5′–3′ orientation: Id2f/f (forward) 5′-TGTGCATAATTAATCGCATCA-3′ and (reverse) 5′-TTGGGAAGTCACATTTGTAGTG-3′; Id3f/f (forward) 5′-GCTCTGAGGTCATAAATCCC-3′ and (reverse) 5′-CCATTTGGTTCTATGTATGCCCGTG-3′; LckCre (forward) 5′-GCAGGAAGTGGGTAACTAGACTAAC-3′ and (reverse) 5′-TCTCCCACCGTCAGTACGTGAGATATC-3′; CD1d WT (forward) 5′-AGGGCTGTGTAGAACTCTGGCGCTA-3′ and (reverse) 5′-GCAGGGAGCGGAAGGTGTAATT-3′; and CD1d KO (forward) 5′-AGGGCCAGCTCATTCCTCCACT-3′ and (reverse) 5′-GCAGGGAGCGGAAGGTGTAATT-3′. The following real time PCR primers were used: IL-4 (forward) 5′-ATCATCGGCATTTTGAACGAGGTC-3′ and (reverse) 5′-ACCTTGGAAGCCCTACAGACGA-3′.

Cell sorting and microarray

Premalignant iNKT cells were sorted as TCRβ+CD1dTet+ cells from L-DKO mice at 20 d of age. Lymphoma cells were sorted from tissues of 18−37-wk-old mice as T cells that are CD1dTet+ or CD1dTet−. Total RNA was extracted as described for real time PCR. mRNA expression profiling was done by the Duke Microarray Core Facility using GeneChip Mouse Genome 430A 2.0 arrays (Affymetrix). The microarray data have been submitted to the Gene Expression Omnibus repository under the accession number GSE83761.

Bioinformatics and statistical analysis

Microarray data for premalignant and lymphoma cells were normalized using RMA, and differential analysis was done using the limma package available through Bioconductor (45). Publicly available normalized ImmGen data for WT cells were requested and downloaded from http://rstats.immgen.org/DataRequest/ (46). The two normalized datasets were combined according to the Empirical Bayes method using the Web tool ArrayMining (http://www.arraymining.net) (47).

Data plotting, visualization, and statistics

Gene-expression (fold change) heat maps were generated using Gene-E (http://www.broadinstitute.org/cancer/software/GENE-E/). Self-organizing maps (SOM) were generated by the Partek Genomics Suite made available by the Duke Center for Genome and Computational Biology. Principal component analysis was performed and plotted using the built-in R functions prcomp and plot3d in open-source RStudio software. Gene overlaps in the form of a Venn diagram were drawn using eulerAPE software (48). Pathway analysis was done using HOMER software (49). Survival curves and bar graphs were drawn using GraphPad Prism (GraphPad). The two-tailed Student t test was used for statistical analyses, with p values < 0.05 considered significant.

Results

Conditional deletion of Id2 and Id3 leads to rapid lymphoma development

We previously reported a dramatic expansion of iNKT cells upon Id2/Id3 conditional deletion using LckCre in L-DKO mice (34). Surprisingly, we found that these mice also rapidly develop tumors and start dying between 3 and 11 mo of age (Fig. 1A). L-DKO mice developed lymphoma in several organs, including thymus, lymph nodes, bone marrow, spleen, liver, and gut (Table I). Splenomegaly and hepatomegaly were apparent in all mutant mice analyzed in this age window, which also were reflected in the significant increase in the weight of liver and spleen of these mice compared with control mice (Fig. 1B). Histopathological H&E staining of the thymus, spleen, liver, lung, and kidney of L-DKO tumor mice revealed infiltration of lymphoma cells in these organs, as well as the disruption of normal tissue structures (Fig. 1C). Masson’s trichrome staining, which is used to detect collagen and fibrosis in tissues, suggested a mild fibrosis in the lymphoma-infiltrated area of livers of L-DKO mice aged ≥5 mo (Fig. 1D). At this stage, the normal liver parenchyma was replaced by malignant lymphocytes, which could also lead to liver dysfunction and death. Thus, we observed that conditional deletion of Id2 and Id3 led to lymphocyte infiltration and rapid development of tumors in various peripheral organs, which ultimately caused their death.

FIGURE 1.
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FIGURE 1.

Deficiency of both Id2 and Id3 in developing T cells leads to lymphomagenesis in mice. (A) Survival curve for L-DKO and control (LckCre−) mice. The p value was determined using the Mantel–Cox test. (B) Comparison of size and weight of spleen and liver from L-DKO (n = 5) and WT control (n = 4) mice. (C) Representative H&E staining for thymus, spleen, liver, lung, and kidney from L-DKO mice and controls (original magnification ×100). (D) Representative Masson’s trichrome staining for livers from L-DKO mice and controls at 2, 4, and 5 mo of age (original magnification ×400). n = 3 for (C) and (D). **p value between 0.001 and 0.01, ***p value between 0.0001 and 0.001.

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Table I. Phenotypic characteristics of tumors in L-DKO mice

L-DKO lymphomas are derived from CD1dTet+ iNKT cells or CD1dTet− innate-like T cells

Because we previously observed neonatal expansion of iNKT cells in L-DKO mice, we hypothesized that the tumors in these mice are derived from the uncontrolled expansion of the iNKT cell population. However, upon further assessment, we found that only 36% of L-DKO mice developed CD1dTet+TCRβ+ tumors, whereas the rest primarily developed CD1dTet−TCRβ+ tumors (Fig. 2A, Table I).

FIGURE 2.
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FIGURE 2.

Lymphomas in L-DKO mice are CD1dTet+ (iNKT) or CD1dTet− in origin. (A) Representative staining of thymocytes with CD4 and CD8 markers from a Cre− control and two L-DKO mice with CD1dTet+ (L60) or CD1dTet− (LIII10) tumor. CD4 and DN fractions were analyzed further by staining with TCRβ and CD1dTet without or with loaded Ag. (B) Representative graphs of intracellular PLZF staining of TCRβ+ populations from mice (n = 2) with tumor shown in (A). (C) Detection of Vα14Jα18 rearrangement in CD1dTet+ or CD1dTet− lymphoma samples by PCR. CD14 was used as a loading control (n = 5).

A recent publication from Cornelis Murre's group has also described the expansion of innate-like follicular helper T cells and lymphoma development in Id2f/fId3f/fIL7RCre mice (50). To better characterize the lymphoma populations in L-DKO mice (with a later deletion of Id2/Id3), we started by examining the expression of PLZF, a key transcription factor for all innate-like cells, including iNKT cells (29, 51). High PLZF expression in the CD1dTet+TCRβ+ population verified that these were indeed NKT cells (Fig. 2B, upper panels). The NKT lymphoma cells were further confirmed to be iNKT cells by their typical Vα14Jα18 rearrangement, which was not observed in CD1dTet− cells (Fig. 2C). Interestingly, CD1dTet−TCRβ+ cells also were found to be PLZF+, distinguishing them from conventional αβ T cells (Fig. 2B, lower panels). Phenotypic analysis of tumor samples from several L-DKO tumor mice also revealed that the majority of lymphoma cells (both CD1dTet+ and CD1dTet−) expressed the surface markers CD69 and CD44 but lacked CD25 and CD24 expression, similar to innate-like iNKT cells (Table I). These expression patterns further verified that the PLZF+CD1dTet−TCRβ+ population was innate-like. Although other groups of investigators also reported the expansion of PLZF+CD1dTet−TCRβ+ populations in the absence of Id proteins, the different stage of Id deletion and lack of comprehensive surface markers make these populations difficult to compare (52, 53). Thus, we found that L-DKO mice develop innate-like T cell lymphomas that are derived from CD1dTet+ iNKT cells or CD1dTet− innate-like T cells.

Malignant innate-like T cells from L-DKO mice are able to invade healthy tissues

To evaluate the tumorigenic potential of innate-like lymphoma cells in L-DKO mice, we transferred these cells into Rag2−/− mice. We found that 70% of the Rag2−/− recipients died within 7–12 wk of transfer of L-DKO lymphoma cells (Fig. 3A). Substantial lymphocyte infiltration was observed by H&E staining of liver and spleen tissues from recipient mice (Fig. 3B). It was also evident that CD1dTet+ cells and CD1dTet− cells were capable of giving rise to secondary lymphomas in Rag2−/− hosts. These secondary lymphomas matched the original phenotype of the donor innate-like lymphoma cells, such that lymphomas derived from donor 1 cells were CD1dTet+PLZF+, whereas those from donor 2 gave rise to CD1dTet−PLZF+ lymphomas (Fig. 3C). Adoptive transfer of lymphoma cells into WT hosts also gave rise to tumors within 10 wk (Supplemental Fig. 1), indicating that these tumors acquire the ability to evade immune surveillance. These results demonstrated the malignancy of these innate-like lymphomas derived from L-DKO mice.

FIGURE 3.
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FIGURE 3.

Adoptive transfer of L-DKO lymphoma cells gives rise to tumor in Rag2-deficient recipients. (A) Survival curve for Rag2−/− hosts after receiving 5 × 106 lymphoma cells from L-DKO mice (n = 10). Rag2−/− mice that were not injected with lymphoma cells were used as control (n = 3). The p value was determined using the Mantel–Cox test. (B) Representative H&E staining for spleen and liver from WT control, L-DKO mice with lymphoma, Rag2−/− control, and Rag2−/− mice that received lymphoma cells (n = 5 for each host type). Original magnification ×100. (C) CD45.2+ lymphoma cells in recipient mice analyzed for their CD1dTet and TCRβ expression. Intracellular PLZF expression levels in CD1dTet−TCRβ+ and CD1dTet+TCRβ+ (iNKT) control cells from Cre− mice and CD45.2+ tumor cells from Rag2−/− recipients. Data are representative of three analyzed recipients.

CD1dTet− innate-like T cells start expanding in neonatal L-DKO mice

Next, we wanted to explore the possible neonatal expansion of these cells in L-DKO mice, similar to iNKT cells. After gating out γδ T cells, which aberrantly upregulate CD4 and CD8 in L-DKO mice, we found that the CD1dTet−TCRβ+ population was indeed expanded in 20-d-old L-DKO mice (Fig. 4A). This population had markedly upregulated PLZF expression, clearly demarcating them from conventional CD4 single-positive (SP) and double-negative (DN) cells. Interestingly, the PLZF level in CD4 SP CD1dTet− cells was even higher than that in iNKT cells. Compared with WT conventional CD4 T cells, we found that innate-like CD4+CD1dTet− cells had a lower surface expression of TCRβ and CD24 that was similar to iNKT cells from L-DKO mice (Fig. 4B). We also looked at surface expression of CD122 and CD25, which are usually upregulated in type II NKT cells. However, we found no significant upregulation of these markers among the L-DKO CD1dTet− population (Fig. 4B). These cells had a broader and more evenly spread TCRβ usage than did CD1dTet+ iNKT cells, which is more typical of type II NKT cells (Fig. 4C). A unique feature of innate-like lymphocytes is their ability to produce IL-4 in the steady-state. Quantitative PCR analysis revealed that the CD4+CD1dTet− population had a higher expression of IL-4 in the steady-state compared with iNKT cells and conventional CD4 T cells, which indicated a possible regulatory role for this subset (Fig. 4D). These observations are indicative of neonatal expansion of the innate-like CD1dTet− population, which has shared features with type II NKT cells.

FIGURE 4.
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FIGURE 4.

Innate-like CD1dTet− T cells expand in the absence of Id2 and Id3. (A) Representative flow cytometry analysis of thymocytes (TCRγδ+ cells gated out) from 20-d-old L-DKO and Cre− control mice. Cells were stained for CD4 and CD8 to separate the CD4+ and DN populations, which were further analyzed with TCRβ and CD1dTet markers. Intracellular PLZF staining is shown for the corresponding CD1dTet− and CD1dTet+ (iNKT) cells from the CD4 SP and DN fractions, along with Cre− DP cells as controls. Absolute numbers of CD4+CD1dTet−TCRβ+PLZF+ cells for 20-d-old L-DKO and Cre− mice (n = 3 for each). (B) Representative surface staining of thymocytes gated on CD4+CD1dTet+ (iNKT) or CD4+CD1dTet−TCRβ+ cells from 20-d-old L-DKO or Cre− control mice. Graphs show TCRβ, CD24, CD25, and CD122 staining (n = 2). (C) TCRβ-chain distribution among CD4+TCRβ+CD1dTet+ (iNKT) or CD4+TCRβ+CD1dTet− cells from 20-d-old L-DKO (n = 4) and Cre− controls (n = 4), as measured by a panel of corresponding Vβ Abs. (D) IL-4 transcript expression in sorted CD4+TCRβ+CD1dTet+ (iNKTs) and CD1dTet− cells from 20-d-old Cre− and L-DKO mice, as measured by real time PCR (n = 4). **p value between 0.001 and 0.01, ***p value between 0.0001 and 0.001.

CD1dTet− cells develop in a CD1d-independent manner and expand to cause lymphoma in L-DKO CD1d-deficient mice

Because we found low expression of CD122 and CD25 but a diverse TCRβ repertoire among L-DKO CD1dTet− cells, we needed to further verify whether the L-DKO CD1dTet− cells belonged to the type II NKT lineage. It is known that all types of αβ NKT cells depend on CD1d-mediated selection for their development (54). Therefore, we generated TKO mice and found that the CD1dTet−TCRβ+ population was still existent in the absence of CD1d (Fig. 5A). PLZF expression also verified that CD1dtet−TCRβ+ cells in TKO mice were innate-like (Fig. 5B). TCRα repertoire analysis indicated that CD1dTet−TCRβ+ cells from TKO and L-DKO mice had fairly broad distribution of TCRα-chains, with no clear preference for Vα14 or Vα3, unlike iNKT cells and type II NKT cells (Fig. 6A). Similar TCRβ usage also suggested that the same CD1dTet− population was present in TKO and L-DKO mice (Figs. 4C, 6B). Interestingly, one of the TKO mice demonstrated a dominant usage of Vβ7, which suggested possible clonal expansion and tumorigenic potential as early as 20 d of age. Cumulatively, these data verified that these CD1dTet− cells are innate-like and a novel type of CD1d-independent NKT cell, similar to γδ NKT cells. We found that lymphocyte infiltration by the expanded CD1dTet− population also gave rise to tumors in TKO mice (Supplemental Fig. 2).

FIGURE 5.
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FIGURE 5.

Expansion of CD1d-independent innate-like T cells in TKO mice. (A) Representative staining of thymocytes from 20-d-old Cre− control, Cre− CD1d−/− control, or L-DKO CD1d−/− (TKO) mice using CD4 and CD8 markers (top panels). CD4+ and DN gated cells were further analyzed for CD1dTet and TCRβ expression. (B) Representative intracellular PLZF staining for TCRβ+ cells from 20-d-old WT (Cre−), Cre− CD1d−/−, and TKO mice (n = 3).

FIGURE 6.
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FIGURE 6.

CD1d-independent CD1dTet−TCRβ+ T cells have broad TCRα and TCRβ repertoires. (A) Vα and Jα repertoires (percentage usage) of CD1dTet−TCRβ+ T cells from 20-d-old TKO mice (n = 2) and L-DKO (n = 1) mice, as measured by 5′RACE (Invitrogen). TRAV11 and TRAV9 correspond to Vα14 and Vα3 chains, respectively, according to the new HUGO Gene Nomenclature Committee. (B) TCRβ-chain distribution among CD4+TCRβ+CD1dTet− cells from 20-d-old TKO mice (n = 4), as measured by a panel of corresponding Vβ Abs. Repertoire for each individual mouse is depicted by a separate pattern.

Neonatal L-DKO iNKT cells have a unique transcriptional program that promotes NKT cell development and expansion and predisposes them to lymphomagenesis

We next sought to identify the dysregulated molecular mechanisms responsible for tumor initiation and development. We did a microarray analysis to compare premalignant iNKT cells from 20-d-old L-DKO mice and lymphoma cells (CD1dTet− or CD1dTet+/iNKT in origin) from L-DKO mice with well-developed tumors. We found that the tumors upregulated several genes and downregulated fewer genes compared with premalignant iNKT cells (Fig. 7A). Hierarchical clustering also demonstrated a clear distinction between lymphoma and premalignant iNKT cells, as well as more variability between the tumor samples (Fig. 7A). To allow direct comparisons of our L-DKO premalignant and tumor cells with control WT NKT cells and WT double-positive (DP) cells, we combined our microarray data with publicly available ImmGen data (46). Clustering patterns showed clear segregation among the WT NKT, L-DKO neonatal, and tumor samples (Fig. 7B, 7C). We found that there were both shared and unique gene signatures among WT NKT cells, premalignant L-DKO iNKT cells, and CD1dTet+ NKT tumors; surprisingly, there was only a moderate overlap between 20-d L-DKO NKT cells and WT NKT cells (Fig. 7B, 7D).

FIGURE 7.
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FIGURE 7.

Aberrant gene-expression program in neonatal NKT cells in the absence of Id2 and Id3. (A) Heat map with hierarchical clustering showing gene expression in sorted neonatal NKT cells from 20-d-old L-DKO mice and tumor cells (CD1dTet+ and CD1dTet−) from 18- to 37-wk-old mice, as measured by mouse genome arrays. Colors represent global values of low (blue) to high (red) gene expression, with values ranging from 2.48 to 14.13 (in log2 scale, normalized values). Age of the mice, tissue origin of cells, and their phenotype are also listed. (B) SOM showing gene expression in clusters of genes for tumor or NKT cells from mice listed above or from WT control mice (combined data). Colors represent low (blue) to high (red) log2 fold change in gene expression with respect to WT DP cells. (C) Principal component analysis for L-DKO NKT, NKT tumor, and CD1dTet− tumor samples [described in (A)], combined with WT NKT and WT DP cells from ImmGen. (D) Venn diagram represents the number and percentage of NKT-specific genes (p < 0.05 and absolute fold change > 2 in WT NKT cells with respect to WT DP cells) that are unique or shared among WT NKT, neonatal L-DKO NKT, and NKT tumor cells. (E) Heat map showing hierarchical clustering and relative log fold change of gene expression in WT NKT and neonatal L-DKO NKT cells with respect to WT DP cells. Genes were selected based on expression patterns of SOM clusters (listed in Supplemental Table I). (F) Heat map showing the relative log fold change for genes involved in cytokine–cytokine receptor interaction. For (E) and (F), colors represent the lowest (blue) to highest (red) fold change of a particular gene among the different samples.

Based on the expression patterns of gene clusters in the samples, we identified genes that were differentially expressed in WT or L-DKO iNKT cells (Fig. 7B, Supplemental Table I). Interestingly, a significant fraction of genes were downregulated in L-DKO iNKT cells compared with WT NKT cells, which included antiproliferative and proapoptotic genes Pawr (55) and Lgals1 (56) and tumor-suppressor genes Cebpb (57) and Irf5 (58) (Fig. 7E). Genes implicated in cell cycle progression and metastasis, such as Vangl2 (Wnt pathway), Cdk1 (p53 pathway), Ccr7, and Igfbp4, were significantly upregulated in L-DKO iNKT cells. Dgka, which was reported to be important for NKT cell development (59), as well as for promoting tumorigenesis (60), was also found to be upregulated >2-fold in L-DKO iNKT cells. These expression patterns supported the tumorigenic potential of these cells.

In contrast, these premalignant cells also demonstrated the upregulation of several cell cycle arrest, tumor-suppressor, and antiproliferative genes, such as Rprm (61), Ptpn14 (62), and Btg2 (63) (Fig. 7E). Other genes that commonly contribute to tumor development or are overexpressed in tumors, such as Pkd2 (64), Mmp2 (65), Adm (66), and Vcam1 (67), had reduced expression in these cells. Genes involved in cytokine–cytokine receptor interaction, many of which were implicated in facilitating tumor metastasis, were also downregulated (Fig. 7F) (68). These data hint toward the existence of a tumor-suppression program in these cells that prevents tumorigenesis at this stage.

Rag2 was found to be upregulated >3-fold in L-DKO iNKT cells, which would allow prolonged TCRα rearrangement to increase chances of the distal Vα14Jα18 rearrangement to promote iNKT cell development (Fig. 7E) (69). We previously described a block in iNKT development beyond stage 1 in L-DKO mice, which allows these cells to constantly proliferate without undergoing maturation (34). We found downregulation of Relb in L-DKO iNKT cells, which was described as being critical for the developmental progression of NKT cells to stages 2 and 3 (70). Overall, gene expression and pathway analysis revealed that Id2/Id3 deletion initiates a modified transcriptional program in L-DKO iNKT cells that supports their prolific expansion while maintaining a premalignant state.

Conditional Id2/Id3 deletion promotes acquisition of multiple tumorigenic programs

Because the tumors in L-DKO mice share lineage identity with iNKT cells or CD1dTet− cells that had undergone persistent expansion starting at a neonatal age, it is likely that the acquisition of secondary and tertiary oncogenic mutations ultimately led to tumor development. We found a clear distinction between the tumor samples that could indicate the existence of multiple, varying aberrant pathways that are reflective of the history of accumulated mutations in the parent cells, which was also supported by their oligoclonal expansion patterns (Fig. 7C). Karyotyping analysis of lymphoma cells from L-DKO mice also revealed aneuploidy in one of two tumor cases (Supplemental Fig. 3). This could be an indication of chromosome instability, which is often linked to tumorigenesis, and may play a role in promoting lymphoma development in L-DKO mice (49, 71).

Because the CD1dTet− tumor sample was significantly distant from CD1dTet+ tumors, it was treated as a unique outlier and was not analyzed further. We then focused our attention on iNKT cell tumor samples and identified genes with >2-fold expression in L-DKO iNKT cells, iNKT tumors, or WT NKT cells compared with WT DP cells (Fig. 7D). Of these genes, iNKT tumor cells had a largely unique gene profile, with only a small fraction retained in common with WT NKT cells and premalignant L-DKO iNKT cells. We also performed pathway analysis to identify the pathways overrepresented in the tumor samples (Fig. 8A) (49). This analysis revealed many aberrant pathways specific to the tumor samples, particularly corresponding to cytokine–cytokine receptor interaction, NF-κB signaling, and transcriptional misregulation in cancer (Fig. 8A, 8B). Interestingly, the cytokine–cytokine receptor pathway, which was downregulated in premalignant iNKT cells, was found to be upregulated upon tumorigenesis. This included genes (Csf1r) that aid in proliferation and can act as proto-oncogenes (72), as well as chemokines and their ligands (Cxcl12, Ccl11, Ccl9, Ccr1, Ccr5) that contribute to metastasis (73, 74). We noted that upregulation of Csf1r was uniquely detected in iNKT cell lymphomas but not in CD1dTet− lymphomas (Fig. 8C). Icam1 and Cxcl12, which are reported to regulate NKT cell homing to the liver and bone marrow (75, 76), were also upregulated in the tumor cells and, therefore, could potentially promote accumulation of iNKT cells and tumor formation (77, 78) in these organs (Table I). Lta and Ltb are known to play roles in iNKT thymic emigration (79) and were significantly upregulated in iNKT tumors. Furthermore, several interesting genes were involved in the transcriptional misregulation of cancer, such as Runx2, Mmp3, Mmp9, Egln3, and Vegfa (80–83). We also found upregulation of the Id2 transcript in iNKT tumors compared with premalignant iNKT cells (Fig. 8B). This could represent overexpression of Id2 exon 3, which is the only remaining exon in Id2f/f mice (84).

FIGURE 8.
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FIGURE 8.

The concurrent loss of Id2 and Id3 turns on an oncogenic program in NKT cells. (A) Pathways overrepresented by genes with >2-fold gene expression in NKT tumor samples according to gene sets annotated by Kyoto Encyclopedia of Genes and Genomes (96). Percentages represent the fraction of genes from each pathway that are overexpressed in the samples. (B) Heat maps showing relative log fold change in gene expression with respect to WT DP cells for genes from pathways identified in the above analysis. (C) Representative CSF1R surface staining for CD1dTet+, CD1dTet− lymphoma samples (n = 4) from L-DKO, TKO, and WT control mice (n = 3). (D) Heat maps showing select genes with significant differential expression among premalignant and tumor samples. (E) Graphic depicting a few key overrepresented pathways in premalignant NKT cells from 20-d-old L-DKO mice (purple) or in CD1dTet+ NKT lymphoma cells from older L-DKO mice (orange). Downregulation (blue), upregulation (red), or partial upregulation and downregulation (yellow) of selected genes from the pathways are also shown. (F) Fold change in gene expression, with respect to WT NKT cells, of genes that are implicated in human NKT tumors (90) and also dysregulated in L-DKO iNKT tumors. Percentages refer to patients with NKT lymphomas (total n = 25) who have mutations in the listed genes, as characterized by Jiang et al. (90).

We then identified interesting genes that were unique to L-DKO iNKT or iNKT tumor samples (Fig. 8D). Genes supporting cell proliferation, such as Cd74 (85) and Adm (66), were downregulated in premalignant iNKT cells but were highly upregulated in iNKT tumors. The tumor suppressor, Pcgf2 (86), was also significantly downregulated in iNKT tumor cells. Interestingly, Ccr7 (87) and Egr2 (88, 89), which were shown to play critical roles in NKT development, were specifically upregulated in premalignant iNKT cells. Therefore, gene-expression profiling revealed several known oncogenic pathways that may contribute to the development of iNKT tumors. This analysis also indicated that there were distinct pathways involved in the premalignant expansion of iNKT cells and the ultimate transformation of iNKT cells leading to uncontrolled tumor growth (Fig. 8E). However, comparison of these identified genes and pathways revealed only a limited overlap with the lymphomas described in Id2f/fId3f/fIL7RCre mice (data not shown). We also found a moderate, but varying, upregulation of CXCR5 in L-DKO lymphoma cells (data not shown) (50). The phenotypic difference between NKT tumors in L-DKO mice and follicular helper T cell tumors in Id2f/fId3f/fIL7RCre mice indicates that tumor types may be dictated by the timing of Id gene deletion.

Additionally, to determine similarity with human NKT tumors, we compared our L-DKO tumor data with a recent publication characterizing key driver mutations and pathways in patients with NKT lymphomas (90, 91). We found that many of the mutated genes in human patients from their study (90) were also dysregulated in the L-DKO tumor model. The shared genes had differential expression patterns in L-DKO iNKT tumor samples and neonatal iNKT cells compared with WT NKT cells (Fig. 8F). The article also implicated upregulation of the NF-κB pathway in driving tumorigenesis in a subset of patients with poor prognosis (90). Several genes of the NF-κB pathway also were found to be uniquely upregulated or downregulated in L-DKO iNKT tumors but not in premalignant neonatal iNKT cells (Fig. 8B). These data suggest that this is a potential mouse model to investigate mechanisms of iNKT and innate-like tumors in humans.

Discussion

A previous study of Id3−/− mice revealed a tumor-suppressor role for Id3 in the development of hepatosplenic T cell lymphoma–like tumors (20). In this study, we observed iNKT cell and innate-like T cell tumors upon deletion of both Id2 and Id3. It is interesting to note the difference in the kinetics of lymphoma development and progression in Id3−/− mice and L-DKO mice. Id3−/− mice often develop autoimmune diseases (92), but tumor development is much more infrequent and delayed, such that these mice live for ≥1 y. In contrast, L-DKO mice start dying of tumor by 3 mo of age. This rapid development of αβ T cell lymphomas in Id2f/fId3f/fIL7RCre+ mice (50) and iNKT cell and CD1dTet− tumors in L-DKO mice argue in favor of Id2 playing novel compensatory and nonredundant roles, together with Id3, in the regulation and suppression of tumorigenesis of developing T cells in the murine thymus.

Previous reports demonstrated a role for Id proteins in suppressing the development of innate-like γδ and iNKT cells (29, 35). Our findings highlight the suppressive role of Id proteins in overall innate-like T cell development, such that there is also expansion of CD1dTet− innate-like T cells in L-DKO mice. Interestingly, this population bears a resemblance to the CD4+PLZF+ cells that expand in mice deficient in Itk or Id3 or with early deletion of both Id2 and Id3 (50, 52, 93). Additional characterization of surface markers and gene-expression programs in these cells would be important for comparing these innate-like T cells and for understanding the cellular origin of iNKT and innate-like T cell tumors in humans (44, 48, 94, 95).

To delve into the mechanism(s) of tumor formation in L-DKO mice, we performed a meta-analysis by combining our L-DKO microarray data with WT data. This allowed us to perform direct comparisons between our mutant cells and WT NKT cells, which were originally not included in our microarray analysis. However, this approach limits the analysis to only the common gene probes in the two microarray datasets, which can lead to the omission of some potentially interesting genes in this model. It is also important to note that the expanded iNKT cells in L-DKO mice are a heterogeneous population consisting primarily of stage 1 and stage 2 NKT cells (34). Because of the lack of availability of an appropriate control population, we used mature NKT cells from B6 mice as reference. Despite this distinction between the populations, it is reasonable to make this comparison as a reflection of changes in transcriptional programs in NKT cells lacking Id2 and Id3 that lead to lymphoma development. With the microarray datasets combined, we were able to observe the deviation in the genetic program in L-DKO iNKT cells compared with WT NKT cells. We found that several cell cycle genes were upregulated and proapoptotic genes were downregulated in neonatal L-DKO iNKT cells. This modified program allowed their dramatic expansion but also kept their tumorigenic potential in check. We inferred that these expanding neonatal innate-like cells are stochastically driven toward tumorigenesis via different pathways, giving rise to heterogeneous tumors in these mice.

Among L-DKO tumors, we observed dysregulation of genes in several pathways, such as transcriptional misregulation in cancer and cytokine–cytokine receptor interactions, as well as others that are commonly overexpressed in various cancer types. Although it is difficult to determine with certainty which genes contributed to lymphoma development versus those that were upregulated as a result of lymphoma development, we verified the sharing of key genes and pathways in L-DKO tumors and human NKT tumors. Therefore, these mice serve as an appropriate mouse model to study iNKT and innate-like tumors. Furthermore, the striking resemblance between all premalignant NKT samples and the divergence of tumor samples leads us to the enticing prospect of treating tumors by identifying and targeting early tumorigenic pathways. Such a study of tumor initiation and gradual progression is only possible in a mouse model and would be useful in determining common genes that lead to malignant transformation.

It is indeed an interesting proposition that Id2 and Id3, through their suppression of innate-like cell fate, prevent unchecked expansion of iNKT cells and CD1dTet− cells under WT conditions. Therefore, upon deletion of Id2 and Id3, the rapid proliferation and expansion of these cells makes them prone to accrual of additional mutations leading to tumorigenesis by various mechanisms.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. A. Lasorella and Dr. A. Iavarone (Columbia University) for sharing the Id2f/f strain. We thank M. Dai (Duke University) for technical assistance with generating the initial L-DKO breeding colony and adoptive transfer of lymphoma cells, the Duke Cancer Center Flow Cytometry Facility for assistance with cell sorting, the Duke Cancer Center Sequencing Facility for assistance with ion torrent sequencing, and the National Institutes of Heath Tetramer Core Facility for providing CD1dTet. We thank S. Sen (Carnegie Mellon University) for assistance with bioinformatics analysis and R implementation.

Footnotes

  • This work was supported by National Institutes of Health Grants R01 GM059638 and P01 AI102853 (to Y.Z.).

  • The microarray data presented in this article have been submitted to the Gene Expression Omnibus repository (https://www.ncbi.nlm.nih.gov/projects/geo) under accession number GSE83761.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CD1dTet
    CD1d tetramer
    DN
    double-negative
    DP
    double-positive
    Id
    inhibitor of DNA binding
    iNKT
    invariant NKT
    L-DKO
    Id2f/fId3f/fLckCre+
    PLZF
    promyelocytic leukemia zinc finger
    SOM
    self-organizing map
    SP
    single-positive
    TKO
    Id2f/fId3f/fLckCre+CD1d−/−
    WT
    wild-type.

  • Received November 14, 2016.
  • Accepted February 7, 2017.
  • Copyright © 2017 by The American Association of Immunologists, Inc.

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The Journal of Immunology: 198 (8)
The Journal of Immunology
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15 Apr 2017
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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 Rajagopalan, Sandeep Dave, Anna Mae Diehl, Yuan Zhuang
The Journal of Immunology April 15, 2017, 198 (8) 3136-3148; DOI: 10.4049/jimmunol.1601935

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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 Rajagopalan, Sandeep Dave, Anna Mae Diehl, Yuan Zhuang
The Journal of Immunology April 15, 2017, 198 (8) 3136-3148; DOI: 10.4049/jimmunol.1601935
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