Notch Activation in Cord Blood Progenitors Induces a CD7+ Common NK/T Precursor Capable of NK Cell Commitment without Full Maturation and Differentiation of Lineage Committed Pre-T Lymphocytes with a Th1 Phenotype.

Activation of Notch signaling regulates differentiation and homeostasis of hematopoetic stem cells. After stimulation, intracellular Notch is proteolytically released and by binding the CSL complex and co-activator MAML, and initiates transcription of downstream genes. We hypothesize that Notch is important for distinct stages of lymphoid development. Human cord blood CD34+ progenitor cells were transduced with retrovirus based eGFP-control, eGFP-Notch and Notch Dominant Negative/MAML (eGFP-DN) constructs. CD34+/eGFP+ were sorted and then co-cultured with the mouse embryonic liver cell line EL08.1D2 and exogenous human cytokines (IL-3. IL-7, IL-15, Flt3 ligand and c-kit ligand). As early as 48 hours after transduction, CD34+/Notch+ cells gave rise to population of lymphoid precursors CD34+CD7+CD10- (42±5% of all cells) while essentially no cells with this phenotype were detected with the control or DN construct. Proliferation of eGFP-Notch transduced cells in a 6-day thymidine incorporation assay was higher compared to eGFP-DN transduced cells (8410±839 vs. 1103±209 cpm; n=3; p=0.00005). Within 7 days 11±1.5% NK emerged from CD34+/Notch+cells compared to 0.8±0.2% of CD34+/eGFP+ control cells (n=5, p=0.0001). NK cell generation peaked at day 28 with a significantly higher expression of CD7 on NK cells (Notch: 75±5% vs. eGFP: 4.5±1%, n=5, p=0.00004), and no B lymphocytes were seen. Analysis of Notch induced NK cells demonstrated early expression of L-selectin and increased expression of CD45RA on all lymphoid progenitors. At 4 weeks, functional testing revealed reduced cytotoxicity against K562 (Notch: 37±0.5% vs. eGFP: 63.5±1.3%; n=7, p=0.007) suggesting immature function. CD34+/Notch+ derived NK lymphocytes also showed diminished acquisition of the lectin-type receptor NKG2A (Notch: 8.3±3% vs. eGFP: 27.4±4.5%; p=0.04) and killer immunoglobulin receptors (Notch: 2.2±0.5% vs. eGFP: 10.8±4: p=0.05). We next asked whether the Notch induced CD7+ precursor was NK restricted or a common NK/T cell precursor. After 5 weeks in culture, a distinct population of CD3+ T-cells emerged (Notch: 18±5% vs. eGFP: 1.6±0.2; n=5, p<0.001%) which were CD4 and CD8 negative and did not express surface TCR a/b or g/d, but expressed high levels pre-T-alpha mRNA. These Notch activated cells were bona fide T-cells based on their capacity to produce IL-2 after PMA/Ca/I stimulation (62.4±4% by intracellular staining) while essentially no IL-2 production occurred from eGFP control cells (1.6±0.2%; p<0.0001). T-cell development was dependent on both Notch and the EL08.1D2 as no T-cells resulted from CD34+/eGFP-Notch in the absence of stroma. These findings suggest that in addition to Notch and exogenous cytokines, other soluble factors are required for T cell development. In conclusion, our data showed that activated Notch pathway leads to differentiation of a common CD7+ lymphoid precursor capable of both early NK cell and T-cell differentiation. This suggests that differences in Notch ligands in local microenvironments (marrow, thymus, lymph node) may be an important mechanism to orchestrate NK and T cell development.