mCD34⁺ cells develop into T/NK progenitors on DLL1/4 interaction. mCD34⁺ cells were obtained from 3 donors, V1 to V3. (A) Expression of indicated T-lineage–associated markers has stabilized after 4 weeks of culture on TSt-4-hDLL1/4 monolayers. For V2 and V3, the population percentage distribution of each marker combination falls within the following ranges: 88% to 92% for CD45RA⁺iCD3⁺ and 45% to 55% CD7⁺CD5⁺. For V1, these percentages are 63% to 68% and 37% to 58%, respectively. (B) #Numbers/donor represent minimum and maximum of fold increases observed in 2 or 3 independent experiments/donor. (C) DNA was isolated from cells cultured on TSt-4 and TSt-4-hDLL1/4 for polymerase chain reaction analysis of gene rearrangements at the TCR locus. At the δ-locus, all donors show clear Dδ-Jδ rearrangement products, whereas Vδ-Jδ rearrangement products are faint and therefore must have occurred less frequently. In contrast to positive controls, 2 products are amplified from DNA of mCD34⁺ cell–derived progenitors. Sequence analysis demonstrated that the large products (> 1000 bp) are partial rearrangements of the δ-locus. (D) When differentiated cells were cultured for another 4 weeks on TSt-4 without DLL, the population lost surface expression of the T-lineage–associated marker CD5. (E) Instead, cells gained expression of the NK-lineage–associated markers NKp46 and NKG2A but did not express the killer cell immunoglobulin-like receptor proteins CD158a-c. Again, there are no differences between hDLL1- or hDLL4-derived cells. Similar results were obtained with another donor. (F) TSt-4–derived cells have killing capacity when coincubated at indicated effector (e) target (t) ratios with the HLA class I–negative leukemia cell line K562. Each point represents the average of a triplicate analysis. Similar results were obtained with another donor. Purified peripheral blood (PB) NK cells from healthy controls always served as positive control.