Id2 Collaborates with Id3 To Suppress Invariant NKT and Innate-like Tumors

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.

Figure 1. Deficiency of both ID2 and ID3 in developing T cells leads to lymphomagenesis in mice

(A) Survival curve for Id2^f/fId3^f/fLCKCre⁺ (L-DKO) and control (LckCre⁻) mice. p value based on the Mantel-Cox test (B) Comparison of size and weight (N = 4 for WT, N = 5 for L-DKO) of spleen and liver from L-DKO and wild type control mice (C) Representative H&E staining for thymus, spleen, liver, lung and kidney from L-DKO mice and controls (100X). (D) Representative Masson’s trichrome staining for livers from L-DKO mice and controls at 2, 4 and 5 months of age (400X). N = 3 for (C) and (D).

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 either CD1dTet⁺ (L60) or CD1dTet⁻ (LIII10) tumor. CD4 and DN fractions were further analyzed by staining with TCRβ and CD1dTet without or with loaded antigen. (B) Representative histogram 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)

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). p value based on 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). (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 are shown. Data is representative of 3 analyzed recipients.

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 day 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 are shown for 20 day old L-DKO and Cre⁻ mice (N=3 for both Cre⁻ and L-DKO mice). (B) Representative surface staining of thymocytes gated on CD4⁺ CD1dTet⁺ (iNKT) or CD4⁺CD1dTet⁻TCRβ⁺ cells from 20 day old L-DKO or Cre⁻ control mice. Histograms are shown for TCRβ, CD24, CD25 and CD122 staining (N = 2). (C) TCRβ chain distribution among CD4⁺TCRβ⁺CD1dTet⁺ (iNKT) or CD4⁺TCRβ⁺CD1dTet⁻ cells from 20 day old L-DKO (N = 4) and Cre⁻ controls (N = 4) as measured by a panel of corresponding Vβ antibodies. (D) IL-4 transcript expression in sorted CD4⁺TCRβ⁺CD1dTet⁺ (iNKTs) and CD1dTet⁻ cells from 20 day old Cre⁻ or L-DKO mice, as measured by RT-PCR (N = 4).

Figure 5. Expansion of CD1d-independent, innate-like T cells in TKO mice

(A) Representative staining of thymocytes from 20 day old Cre⁻ control, Cre⁻ CD1d^−/− control or L-DKO CD1d^−/− (TKO) mice using CD4 and CD8 markers (top panel). CD4⁺ and DN gated cells were further analyzed for CD1dTet and TCRβ expression. (B) Representative intracellular PLZF staining for TCRβ⁺ cells from 20 day old WT (Cre⁻), Cre- CD1d^−/− and TKO mice (N = 3).

Figure 6. CD1d-independent CD1dTet⁻TCRβ⁺ T cells have broad TCRα and TCRβ repertoires

(A) Vα and Jα repertoires (% usage) of CD1dtet⁻TCRβ⁺ T cells from 20 day 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 day old TKO mice (N = 4) as measured by a panel of corresponding Vβ antibodies. Repertoire for each individual mouse is depicted by a separate pattern.

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 day old L-DKO mice and tumor cells (CD1dTet⁺ and CD1dTet⁻) from 18–37 week 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 is also listed. (B) Self-organized map (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 (PCA) 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 greater than two in WT NKT with respect to WT DP) that are unique or shared between 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 Supplementary 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.

Figure 8. The concurrent loss of ID2 and ID3 turns on an oncogenic program in NKT cells

(A) Pathways overrepresented by genes with greater than 2 fold gene expression in NKT tumor samples according to gene sets annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG)^[97]. Percentages represent fraction of genes from each pathway that are overexpressed in the samples. (B) Heat maps showing relative log fold change of 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 day 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 is 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 in top panel indicate the percent of patients with NKT lymphomas (total N = 25) that have mutations in the listed genes, as characterized by Jiang et al.^[90]