In vivo NK cell differentiation assay

MISTRG mice were generated by Regeneron Pharmaceuticals as described (36). This study used an improved version of the original MISTRG mice, where human SIRPA is expressed not as a transgene but as a knock-in allele (34). For in vivo experiments, female MISTRG mice between 18 and 22 weeks of age were used. For analysis of CD39 expression on tissue-residing NK cells in the humanized mouse model, newborn MISTRG mice were transplanted with 5 × 10⁴ human CD34⁺ cells and isolated from umbilical cord blood, as described previously (36), without any preconditioning by irradiation. After 3 to 4 months, the uterus was harvested for analysis. All mouse experiments were performed in accordance with protocols approved by the Linköping Animal Experimentation Ethics Committee. FACS-sorted dNK cells from several donors, ranging from 5 × 10⁵ to 1 × 10⁶ cells (depending on yield, yet same amount per subset) were pooled in 300 μl of PBS and injected intravenously into female MISTRG mice. Control mice were injected with PBS only. IL-15 was incubated for 20 min with IL-15Rα–Fc (both R&D Systems; see table S5) and administered on days 0 and 5 intraperitoneally at 2.5 and 7.5 ng per mouse, respectively. Mice were sacrificed on day 7, and organs were harvested. The liver and spleen were passed through a 100-μm mesh with a syringe plunger and mononuclear cells extracted via Ficoll separation. Marker expression was analyzed in relation to the specific experiment and gates adjusted accordingly.