Extended Data Fig. 3: Impact of IRF8 deficiency on early human hematopoiesis and T cell development.
a,b, Flow cytometric identification of CD326−CD56+ EMPs that were generated from IRF8+/+ and IRF8-/- induced pluripotent stem cells (a), and their frequency (n = 6 per genotype) (b). c, Flow cytometric analysis to determine the impact of IRF8 loss on the generation of TNFR2+CD123+CD127+ hematopoietic progenitors. d-h, Scheme illustrates the collection of conditioned medium from EMO cultures of different conditions at the indicated time points (d). sTNF was measured using flow cytometric bead-based immunoassay in which a standard curve was generated (e). Concentration of sTNF detected in the conditioned medium (n = 6 per condition) (f). The fraction above the bars denotes the actual n from each condition that had detectable sTNF. Flow cytometric analysis of EMOs (n = 4 per condition), cultured with fresh or conditioned medium from day 7 onwards, to identify TNFR2+CD123+CD127+ hematopoietic progenitors (g) and their frequency (h). i,j, Frequency (i) and cell count (j) of CD7+CD5+ T cell precursors (n = 3) that were generated from tmTNF-expressing CD123+CD127+HLA-A2+ progenitors in the ATO-spiking experiment. k,l, Frequency (k) and cell count (l) of CD7+CD5+ T cell precursors (n = 3) that were generated from HLA-A2− HSPCs in the ATO-spiking experiment. Data are representatives of six (a), three (c) and two (g) independent experiments. Data are presented as mean±s.d. of six (b), two (h) and one (i-l) independent experiments. Data are presented as mean of two independent experiments (f: WT). Data are analyzed by two-tailed paired t test (b,k) and one-way ANOVA with Dunnett’s multiple comparisons test (h). WT, wild type; KO, knockout; MFI, median fluorescent intensity; n, EMP induction (b), pool of 2 EMOs (f,h), pool of 2 ATOs (i,j) and biological replicates (k-l); exact P values are provided in the Source data.
