Extended Data Fig. 2: Impact of low dose IRF8 activity on early human T cell development.

a, Scheme illustrates the downstream independent analyses of empty vector (control)- or IRF8-ERT2-transduced HSPCs that were cocultured with OP9-DLL4 stromal cells. b, Flow cytometric staining of IRF8 expression in empty vector (control)- and IRF8-ERT2-transduced cord blood HSPCs at day 2 post transduction. The transduced cells were not treated with 4-OHT. c,d, Volcano plots depict genes that were differentially expressed in 0 nM (c) and 50 nM (d) of 4-OHT treated IRF8-ERT2-expressing cells compared to the untreated control. Genes with a FDR of <0.05 and a log₂ fold change of differential expression of >1 or < -1 were considered significant. e, RNA-seq revealed changes in expression of Notch target genes in empty vector (control)- and IRF8-ERT2 (treated with 50 nM 4-OHT)-expressing CD7⁺ cells from matched donors (n = 3). The normalized counts are CPM. f, The enrichment scores of IFN-related gene signature for the previously annotated populations of human CD34⁺ thymocytes. g,h, Transcription factor motif analyses for the chromatin regions that are significantly and uniquely opened (g) or closed (h) in IRF8-ERT2-expressing CD7⁺ cells that were treated with 50 nM of 4-OHT. i,j, Flow cytometric analysis to examine the expression of tmTNF and TNFR1 during in vitro human early T cell development (i) and their expression patterns (n = 3) were quantified proportionally (j). Data are representatives of two (b) and one (i) independent experiments. Data are presented as mean±s.d. of one experiment (j). Motif enrichment was determined by HOMER algorithm⁹³ that uses ZOOPS scoring coupled with the hypergeometric or binomial enrichment calculations (g,h). Data are analyzed by two-way ANOVA with Šídák’s multiple comparisons test (j). FDR, false discovery rate; CPM, counts per million; TF, transcription factor; n, biological replicates (e,j); exact P values are provided in the Source data.

Source data