Fig. 4
NK cell differentiation from HSPCs to mature effector cells. (A) Gating strategy allowing to distinguish the different stages of NK cell maturation: HSC (CD34 + CD38-), CLP (CD34 + CD38 + CD10 + CD45RA+), immature NK cells (stage 3: CD34- CD45RA + CD56- CD16-), and mature NK cells (stage 4: CD34- CD45RA + CD56bright CD16-, stage 5: CD34- CD45RA + CD56dim CD16 + and stage 6: CD34- CD45RA + CD56dim CD16 + CD57 + KIR2D+/-). Plots at two weeks of differentiation. (B) Tree representation of SPADE clustering colored by pseudotime calculated using trajectory analysis with Wishbone algorithm of NK cell differentiation taking CD34 + CD38- HSC population as starting cell type. The size of nodes is proportional to the number of cells in the given cluster. Calculation and figure were made with the OMIQ software (https://www.omiq.ai/). The differentiation stages have been manually added to the figure in base of the expression of main lineage markers. The same SPADE tree is represented in the lower panel colored based on CD34 and CD56 intensity. For other markers, see Supplemental Fig. 2B. (C) Evolution of NK cell differentiation stages in percentage at two and four weeks of AMO culture (N = 2 at 2 weeks and N = 3 at 4 weeks), calculated as percentage of total NK cells using the gating strategy above mentioned. (D) Phenotype of a representative AMO after two weeks; plot of live NK cells gated on stage 5 cell subset as defined in (A). The positivity of NK cell receptors is assessed by comparison to unstained cells gated on forward scatter. (E) Evaluation of NK cell polyfunctionality after 3 weeks of AMO culture (N = 4). After organoid dissociation, NK cells were cultured either in presence of the NK sensitive K562 cell line or with PMA-Ionomycin. NK cell degranulation of cytolytic granules (surface expression of CD107a) and IFN-γ and TNF-α intracellular richness were determined by flow cytometry. Proportion of NK cells with monofunction, bifunction and trifunction significantly increased after stimulation by K562 cell line or PMA and ionomycin (p-value of 0.02 and < 0.01 for K562 and PMA-Ionomycin versus control respectively, Fisher exact test).
