Characterization of Two Distinct Lymphoproliferative Diseases Caused by Ectopic Expression of the Notch Ligand DLL4 on T Cells

Notch signaling is essential for the development of T cell progenitors through the interaction of NOTCH1 receptor on their surface with the ligand, Delta-like 4 (DLL4), which is expressed by the thymic epithelial cells. Notch signaling is quickly shut down once the cells pass β-selection, and CD4/CD8 double positive (DP) cells are unresponsive to Notch. Over the past two decades a number of papers reported that over-activation of Notch signaling causes T cell acute lymphoblastic leukemia (T-ALL), a cancer that prominently features circulating monoclonal CD4/CD8 double positive T cells in different mouse models. However, the possible outcomes of Notch over-activation at different stages of T cell development are unknown, and the fine timing of Notch signaling that results in T-ALL is poorly understood. Here we report, by using a murine model that ectopically expresses DLL4 on developing T cells, that the T-ALL onset is highly dependent on a sustained Notch activity throughout the DP stage, which induces additional mutations to further boost the signaling. In contrast, a shorter period of Notch activation that terminates at the DP stage causes a polyclonal, non-transmissible lymphoproliferative disorder that is also lethal. These observations resolved the discrepancy of previous papers on DLL4 driven hematological diseases in mice, and show the critical importance of the timing and duration of Notch activity

The Notch pathway is upregulated in DP cells of Tg8 mice.

<p><b>A</b>) Notch1 receptor surface staining of Tg8 DP, CD4 SP and WT DP in thymus (thy) or WT CD4⁺ T cells in spleen (spl). <b>B</b>) Notch intracellular domain (NICD) staining of Tg8 DP, CD4 SP and WT DP in thymus (thy) or WT CD4+ T cells in spleen (spl). <b>C</b>) Thymic DN, DP and SP populations from Tg8 mice and WT littermates were sorted and cDNA was prepared for real time PCR analysis of the indicated genes. Summary of quantitative real time PCR data from three independent sorting experiments. Expression: - <5; +/- 5-15; + 15-50; ++ 50-100; +++ >100, normalized on β-actin expression fixed at 10,000. The yellow shade highlights the DP populations, which display the largest differences between WT and Tg8 mice. <b>D</b>) 5x10⁶ DP or SP cells were sorted from the same Tg8 mice at the early tumorigenic stage, and injected to nude mice. Survival rates of the recipients were recorded in the following 5 weeks. <b>E</b>) NICD staining of Tg8 DN, DP, SP and WT DN cells in thymus and spleen.</p

Tg8 mice develop T-ALL due to over-expression of Dll4 gene on T cells.

<p><b>A</b>) Enlarged hematopoietic organs (spleen, mesenteric lymph node and thymus) but not non-hematopoietic organ (kidney) of 4 month-old Tg8 mice compared with WT (left panel). Survival curve of Tg8 mice compared to WT and Tg5 mice (right panel). <b>B</b>) No obvert aneuploidy in Tg8 lymphomas. On the right panel, a metaphasic spread of one lymphoma; the left panel shows the chromosomes cut from the right side picture and positioned according to their size. Representative of 10 mice. <b>C</b>) Position and orientation of <i>TCRα</i> transgene and Dll4 gene in mouse Chromosome 2. <b>D</b>) Northern blot of Dll4 mRNA expression in Tg8 and WT thymocytes. The same membrane was reblotted with a probe of β actin as a loading control. <b>E</b>) qPCR analysis of Dll4 mRNA expression in Tg8 pre-tumoral thymus, spleen, mesenteric lymph nodes (mLN), skin, heart, kidney, liver and brain compared to WT mice. The abundance is calculated as the relative transcription normalized to β-actin expression fixed at 10,000 (left). <b>F</b>) DLL4 expression is restricted to αβ T cells. Flow cytometry analysis of DLL4 surface expression co-stained with anti-<i>αβ</i> TCR (<i>αβ</i> T cells), anti-<i>γδ</i> TCR (γδ T cells), anti-F4/80 (macrophages), anti-CD11c (dendritic cells), anti-CD19 (B cells) and anti-NK1.1 (NK cells), obtained from spleens of 4 week-old pre-tumoral Tg8 mice and WT littermates. Representative of 5 mice. </p

Characterization of Tg8 cells.

<p><b>A</b>) Pre-tumoral Tg8 and WT thymus have similar distribution of the major lymphocyte populations (DN, DP, CD4 SP and CD8 SP). Representative of more than 20 mice. <b>B</b>) Surface DLL4 staining of the major thymic populations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084841#pone-0084841-g001" target="_blank">Figure 1F</a> (left) and mRNA level of Dll4 in DN, DP and SP thymocytes calculated by real-time PCR (right). <b>C</b>) Pre-tumoral spleens from Tg8 mice accumulate CD4⁺CD8⁺ DP cells. Shown are representative spleens from 4 week-old Tg8 and WT mice. <b>D</b>) Normal splenic architecture in pre-tumoral 3 week-old Tg8 mice. Low power resolution confocal images of Tg8 (top) and WT (bottom) frozen spleen sections. The white pulp can be identified by T cell staining (anti-CD4 and anti-CD8) and B cell staining (anti-B220), and the surrounding red pulp appears in dark. White bar = 100 μm. <b>E</b>) High power resolution of the PALS area of the spleen. Arrowheads point to some of the DP cells. White bar = 6.25μm. <b>F</b>) Peripheral T cells in Tg8 mice, but not WT mice, express surface DLL4. CD4 SP, CD8 SP and DP cells, gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084841#pone-0084841-g002" target="_blank">Figure 2C)</a>, were stained with anti-DLL4. Representative of more than 20 mice. </p

blockade of Notch signaling rescued Tg8 from developing T-ALL.

<p><b>A</b>) Dll4 shRNA delays the onset of Tg8 lymphoma. The appearance of DP cells was monitored by blood staining every 10 days. Data are presented as the change in the DP/CD4 SP ratio over time. n=3 mice per group. <b>B</b>) Conditional KO of γ-secretase in T cells eliminates Tg8 lymphomas. Survival curve of Tg8 PS KO (PS1^fl/fl PS2^-/-) CD4-Cre mice (or PS1^fl/+ as control) (n=10 animals per group). The survival curve of 10 Tg8 mice was also recorded. Note that one PS1^fl/fl mouse escaped complete PS1 deletion (as shown in panel C) and developed lymphoma. <b>C</b>) Efficiency of CD4-Cre-mediated PS1 deletion in different purified thymic and splenic cell populations. TCL: DNA from the only T cell lymphoma observed in the Tg8 PS KO CD4-Cre group. Arrowhead: non-deleted PS1 allele (Floxed). </p