Fig 2: Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs.A, experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP+hNGFR+ LP cells were sorted, and subjected to QuantSeq 3' mRNA sequencing. B, GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs (p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1-deficient LPs are shown. The full lists of the DEGs in Lmo2-, Zbtb1-, Cbfa2t2-, or Cbfa2t3-deficient LPs are shown in Table S2. Data are based on three independent biological replicates. C, heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages (10) in response to the deletion of Lmo2, Zbtb1, or Cbfa2t3. Data are based on the average of three biological replicates. D, five days after sgRNA introduction, GFP+hNGFR+ LP cells were sorted (A). Relative expression levels of Tcf7, Bcl11b, Bcl11a, and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. **p < 0.01, *p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million.

Fig 3: Introduction of Tcf7 rescues the generation of CD25+cells in Zbtb1-deficient LPs.A, experimental scheme for the transduction of Tcf7 after the deletion of Zbtb1 in Ebf1-deficient LPs is shown. B, two days after Tcf7 (encoding TCF1 protein) transduction (A), GFP+CFP+hNGFR+ cells were gated and analyzed for intracellular TCF1 expression. Data are representative of two independent experiments. C, Cas9-GFP and sgRNA-CFP-transduced LPs were introduced with Tcf7-hNGFR, and transferred onto OP9-DLL4 stromal cells for 2 days. GFP+CFP+hNGFR+ cells were gated and analyzed for CD44 and CD25 expression. Data are representative of three independent experiments. D, the percentage of CD25+ cells among GFP+CFP+hNGFR+ cells (C) is shown with SD. The data represent the mean values of three independent biological replicates. **p < 0.01 by two-sided Student’s t test. E, fourteen days after Notch stimulation (A), GFP+CFP+hNGFR+ cells were gated and analyzed for intracellular TCRß expression. Data are representative of two independent experiments. sgRNA, single-guide RNA.

Fig 4: Analysis of transcriptome profile and functions of PD-1Ab21-induced naïve-like T cells.a, b RNA-seq was performed in sorted TN, activated CD44highCD62Lhigh, PD-1Ab21-generated CD44lowCD62Lhigh and IL-15 or IL-2-differentiated cells. a Principal component analysis (PCA) of T cell subsets based on the global transcriptome (~10,000 genes), (n = 3 samples for each cell population). b Heatmap showing the z-transformed expression values of library size-normalized read counts for each differentially expressed gene, colored from blue to red. RNA-Seq data from each independent sample are shown in the columns. Comparisons of gene expression between each pair of CD8+ T cell subsets were done by DESeq2 analysis (Benjamini–Hochberg adjusted P-value < 0.01, fold change > 2). c Flow cytometry analysis for expression of Fas and TCF1 in TN and cells differentiated with different proteins. The isotype (filled histogram) and anti-CD95 (top) or anti-TCF1 (bottom) (open histogram) antibody staining are shown. d Cytokine production of T cells. T cell subsets were sorted as described in a, and stimulated with PMA/ionomycin for 4 h. IFN-? and IL-2 in the supernatants were detected by ELISA. The data are presented as mean ± SEM and are representative of at least three independently performed experiments. Results were compared by unpaired two-tailed t-test. d *p = 0.0165, **p = 0.0019, ***p = 0.0004 (left and right panel), ****p < 0.0001, n.s. not significant.