Notch3 Is Dispensable for Thymocyte β-Selection and Notch1-Induced T Cell Leukemogenesis

Notch1 (N1) signaling induced by intrathymic Delta-like (DL) ligands is required for T cell lineage commitment as well as self-renewal during “β-selection” of TCRβ+ CD4−CD8− double negative 3 (DN3) T cell progenitors. However, over-expression of the N1 intracellular domain (ICN1) renders N1 activation ligand-independent and drives leukemic transformation during β-selection. DN3 progenitors also express Notch3 (N3) mRNA, and over-expression of ligand-independent mutant N3 (ICN3) influences β-selection and drives T cell leukemogenesis. However, the importance of ligand-activated N3 in promoting β-selection and ICN1-induced T cell leukemogenesis has not been examined. To address these questions we generated mice lacking functional N3. We confirmed that DN3 progenitors express N3 protein using a N3-specific antibody. Surprisingly however, N3-deficient DN3 thymocytes were not defective in generating DP thymocytes under steady state conditions or in more stringent competition assays. To determine if N3 co-operates with N1 to regulate β-selection, we generated N1;N3 compound mutants. However, N3 deficiency did not exacerbate the competitive defect of N1+/− DN3 progenitors, demonstrating that N3 does not compensate for limiting N1 during T cell development. Finally, N3 deficiency did not attenuate T cell leukemogenesis induced by conditional expression of ICN1 in DN3 thymocytes. Importantly, we showed that in contrast to N1, N3 has a low binding affinity for DL4, the most abundant intrathymic DL ligand. Thus, despite the profound effects of ectopic ligand-independent N3 activation on T cell development and leukemogenesis, physiologically activated N3 is dispensable for both processes, likely because N3 interacts poorly with intrathymic DL4

Lunatic Fringe Deficiency Cooperates with the Met/Caveolin Gene Amplicon to Induce Basal-Like Breast Cancer

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC

Lunatic Fringe Deficiency Cooperates with the Met/Caveolin Gene Amplicon to Induce Basal-like Breast Cancer

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC

Steady-state thymic immunophenotype and cellularity in <i>N3^LacZ/LacZ</i> mice.

<p>(A) CD4 vs CD8 distribution of total thymocytes from <i>N3^+/+</i> and <i>N3^LacZ/LacZ</i> mice. Thymocytes were stained with anti-CD4-APC and anti-CD8-PE antibodies and analyzed by flow cytometry. (B) CD117 vs CD25 distribution of Lin^- DN thymocytes from <i>N3^+/+</i> and <i>N3^LacZ/LacZ</i> mice. (C) Total thymic cellularity of <i>N3^+/+</i> (black bars) vs <i>N3^LacZ/LacZ</i> (white bars) of 3 week-old (left) and 10 week-old mice (right). Two-tailed Student <i>T</i>-test analyses: <i>P</i> = 1 for 3 week-old <i>N3^+/+</i> (N = 4) vs <i>N3^LacZ/LacZ</i> (N = 4) mice, and <i>P</i> = 0.8 for 10 week-old <i>N3^+/+</i> (N = 4) vs <i>N3^LacZ/LacZ</i> (N = 5) mice.</p

No Evidence for N1-N3 Cooperation during β-selection.

<p>(A) Effect of reduced N1 gene dosage on steady state thymocyte and splenic T cell numbers in <i>N3^LacZ/LacZ</i> mice: Left bar graph shows mean total thymic cell counts from <i>N1^+/+ N3^+/+</i> (grey, N = 3), <i>N1^+/− N3^LacZ/+</i> (white, N = 4), <i>N1^+/+ N3^LacZ/LacZ</i> (hatched, N = 4), and <i>N1^+/− N3^LacZ/LacZ</i> (black, N = 6) mice at 6 weeks of age. Two-tailed Student's <i>T</i>-test comparisons gave <i>P</i> values ranging from 0.2–1 for all pair-wise comparisons. Right bar graph shows average number of TCRβ⁺ splenic T cells at 6–8 weeks of age in <i>N1^+/+ N3^+/+</i> (white, N = 3), <i>N1^+/+ N3^LacZ/LacZ</i> (blue, N = 4), and <i>N1^+/− N3^LacZ/LacZ</i> (black, N = 6) mice. Two-tailed Student's <i>T</i>-test comparisons: <i>N1^+/+ N3^+/+</i> vs <i>N1^+/+ N3^LacZ/LacZ (P</i> = 0.6), <i>N1^+/+ N3^LacZ/LacZ</i> vs <i>N1^+/− N3^LacZ/LacZ</i> (<i>P = </i>0.4), and <i>N1^+/+ N3^LacZ/LacZ</i> vs <i>N1^+/− N3^LacZ/LacZ</i> (<i>P = </i>0.6). (B) N3 does not compensate for lower <i>N1</i> gene dose in <i>N1^+/−</i> thymocytes. Top: Equal mixtures of <i>N1^+/− N3^+/+ CD45.1;CD45.2</i> (black bars) and <i>N1^+/− N3^+/+ CD45.2</i> (white bars) DN3 progenitors co-injected intrathymically into sub-lethally irradiated B6.CD45.1 hosts. One week later, cells from each thymic lobe were stained with anti-CD45.1-PECy7, anti-CD45.2-FITC, anti-CD4-Pacific Blue and anti CD8-PE and analyzed by flow cytometry. The average ratio of CD45.1⁺ CD45.2⁺ to CD45.2⁺ DP progeny was 1.6±0.2. Bottom: Equal mixtures of <i>N1^+/− N3^+/+ CD45.1;CD45.2</i> (black bars) and <i>N1^+/− N3^LacZ/LacZ CD45.2</i> (dashed bars) DN3 progenitors were co-injected intrathymically into sub-lethally irradiated B6.CD45.1 host. One week later, flow cytometry analysis of each lobe shows that the average ratio of CD45.1⁺ CD45.2⁺ to CD45.2⁺ DP progeny was 1.5±0.2. Two-tailed Student <i>T</i>-test comparison of the ratios from each type of competition: (<i>P</i> = 0.5). The experiment was performed three times yielding similar results.</p

N3/LacZ reporter expression in DN3 thymocytes.

<p>(A) Lin- DN thymocytes from N3LacZ/LacZ and N3+/+ mice were loaded with FDG and stained with anti-CD25-APC, anti-CD71-biotin, and anti-CD117-APCCy7 followed by Avidin-PECy5. Top: Left plot shows FSC vs CD25 distribution of CD117- DN thymocytes from N3LacZ/LacZ mice. CD25+ DN3 thymocytes (rectangular gate) were gated to display N3/LacZ vs CD71 (middle) and FSC versus CD71 (right). Rectangles in the middle and right plots show gates used to define DN3a (CD71lo), and DN3b (CD71hi) subsets. Bottom: Histograms show N3/LacZ reporter expression in DN3a and DN3b thymocytes from N3LacZ/LacZ mice (open histograms) compared to background FITC autofluorescence in each N3+/+ subset (shaded histograms). Numbers in each plot depict the average ratio of FITC median fluorescence intensity from N3LacZ/LacZ mice divided by FITC median intensity of the same subset from N3+/+ mice. Numbers in brackets are the standard deviation of this measurement (N = 3 per genotype). (B) Surface expression of N3 protein on DN thymocytes. Open histograms depict anti-N3-PE staining on DN1 (CD117⁺ CD25⁻), DN2 (CD117⁺ CD25⁺), DN3 (CD117⁻ CD25⁺), and DN4 (CD117⁻ CD25⁻) thymocytes from <i>N3^+/+</i> (top), <i>N3^LacZ/+</i> (middle) and <i>N3^LacZ/LacZ</i> (bottom) mice compared to “fluorescence minus one” controls generated by staining <i>N3^+/+</i> thymocytes with all antibodies except anti-N3-PE (shaded histograms). These experiments were repeated twice showing similar results.</p