Fig 2: Tcf1-deficient DN3 cells dedifferentiate into DN1/2-like cells with multipotent lineage capacity.(A) WT DN3 cells sorted and seeded on OP9 WT/OP9-DL1 (10:1) cells develop largely further into DN4 or remain DN3 after 7 days in culture, while Tcf1-deficient cells develop into DN1 and DN2 cells (pregated Thy1+ Lin− cells) with prominent B cell (B220+ CD19+) and myeloid cell (CD11b+ Gr1+) development. (B) Quantification of the developmental plasticity and dedifferentiation effects of DN3 Tcf1-deficient thymocytes into DN1, DN2, myeloid, and B cells. (C) Intracellular TCRβ staining reveals the dedifferentiated DN1 and DN2 cells to be derived from DN3 cells. (Mann-Whitney U test. Error bars represent the SD of three samples from three independent experiments.)

Fig 3: Reexpression of Gata3 suppresses B and myeloid development in Tcf1 deficiency.(A) Layout of retroviral complementation experiments with GFP control and/or Gata3. (B) Gata3 expression partially overcomes the DN1 thymocyte block and (C) suppressed the enhanced non-T cell lineages (B and myeloid cell development) after 7 days in the OP9-DL1 culture system. FACS shows representative plots and graphs quantitative data from replicate measurements. (Multiple t test analysis. Error bars represent the SD from two independent experiments.) (D) In vivo complementation [Ly5.2 Tcf1 (WT or KO)–transduced stem cells transplanted into Ly5.1 recipient mice] reveals suppression of B cell development also in the thymus (right) 8 weeks after transplantation but minimal and partial rescue of T cell development in the thymus (left and middle). (Middle: Multiple t test analysis. Right: Paired t test. Error bars represent the SD from three individual mice per group.)

Fig 4: ß-Catenin partners with TCF-1 to positively regulate EAAT2 at the level of transcription in PDAs. PDAs were transfected with ß-catenin siRNA or Scrm-Si, and 48 h post transfection, cells were transfected with an EAAT2 promoter linked to luciferase, and 24 h later, luciferase activity was measured (a). b PDAs were transfected with pABC or pcDNA, and 24 h post transfection, cells were transfected with EAAT2 promoter linked to luciferase, and 24 h later, luciferase activity was measured. c Identification of putative TCF/LEF binding sites on the EAAT2 promoter. Shown in c is a ~2.5-kb promoter of EAAT2 illustrating TCF/LEF binding sites present in clusters and the regions selected for designing primers for real-time PCR. PDAs were transfected with siRNAs to knockdown TCF-1, TCF-3, TCF-4, LEF1, or scrambled siRNA (d, e), and 72 h post transfection, real-time qPCR was performed to measure the levels of EAAT2 mRNA (d) and protein by WB (e). PDAs (~2 × 106 cells) were cross-linked with 1.0 % formaldehyde and sonicated to generate chromatin preparations (f–h). ChIP was performed using antibodies against ß-catenin (f, g) or TCF-1 (h) (5 µg antibody/IP). PDAs were transfected with EAAT2-prom, and 24 h later, ChIP was run (h). Data shown are normalized to a nontargeting IgG control and reported as a fold change relative to IgG. Asterisks indicate a p < 0.05 relative to the respective control using Student’s t test. Error bars indicate SD

Fig 5: Tcf1/Lef1 TFs control multiple aspects of mature CD8+ T cell functions.a Detection of CCR7 expression with cell-surface staining. Values denote geometric mean fluorescent intensity (gMFI). b T cell homing assay. CTV-labeled CD45.1+ WT and CD45.2+GFP+ dKO CD8+ T cells were mixed at 1:1 ratio (left) and transferred into CD45.2+ recipients, and 24 hrs later, donor cells were detected in recipient LNs (right). Cumulative data on donor cell abundance are means ± s.d. from two experiments. c Detection of EOMES expression with intranuclear staining, with gMFI marked. d Detection of memory-phenotype cells in splenic GFP+CD8+ T cells. Cumulative data on the frequency of CD44hiCD122+ cells are means ± s.d. from two experiments. e, f Detection of MYB expression with immunoblotting (e) and FOXP3 and NRP1 expression with flow cytometry (f). Data are representative from two experiments. g Experimental design for testing CD8+ T cell activation and differentiation in vivo. h–j Detection of CD8+ T cell division and CD25 induction during the first 60 hrs of activation. CTV-labeled splenic CD8+ T cells were transferred into CD45.1+ hosts followed by LCMV-Armstrong infection. CTV dilution and CD25 expression were monitored before (0 hrs), 48 or 60 hrs after infection in recipient spleens. h representative dot plots where dotted lines mark cells in different divisions. i Cumulative data on the frequency of cells in indicated cell divisions at 48 (top) and 60 hrs (bottom) post infection. j. Cumulative data on CD25 gMFI in cells at different dividing stages at 48 (top) and 60 hrs (bottom) post infection. Data are means ± s.d. from two experiments. k Detection of effector P14 CD8+ T cells on day 8 post infection. Effector P14 cells were detected as GFP+CD8+ T cells in recipient spleens, with values in representative contour plots denoting cell frequency. Cumulative data on effector P14 cell counts are means ± s.d. from two experiments. Statistical significance for (b, d, i and k) was assessed with two-tailed Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.