DN3/DN4 transition defects in LKB1-null thymocytes. (A) Flow cytometry of CD44 and CD25 surface expression in CD4⁻CD8⁻ DN thymocytes from LckCre⁺LKB1^fl/fl and LckCre⁺LKB1^+/+ thymi. CD25 and CD44 profiles of Thy-1⁺ DN cells additionally gated to exclude cells from the non-TCR lineage (lineage). Gates show DN1-DN4 populations, representative of five independent experiments. (B) Surface expression of CD25 in lineage Thy-1⁺ thymocytes from LckCre⁺LKB1^fl/fl and LckCre⁺LKB1^+/+ mice, representative of five independent experiments. (C) Genomic PCR analysis of the LKB1 gene in DN3 and CD44⁻CD25^low thymocytes. Top, PCR amplification of the gene product produced only when the LKB1 floxed allele is deleted in Lck-Cre⁺ thymocytes. Bottom, PCR amplification of the non-deleted LKB1 gene. The upper bands represent the PCR product generated from the LKB1 non-deleted floxed gene, while the lower band indicates the WT LKB1 gene. (D) LckCre⁺LKB1^fl/fl CD44⁻CD25^low thymocytes and LckCre⁺LKB1^+/+ DN4 thymocytes were either untreated or pretreated with STO-609 and then stimulated with 50 mM 2-deoxyglucose for 5 min. Western blot of cell lysates prepared from these thymocytes with pThr-172–AMPK and AMPK α1 antisera, representative of three independent experiments.

LKB1 is required for survival of TCRβ selected T-cell progenitors A) DN thymocytes from LckCre⁺LKB1^+/+ (n=3) and LckCre⁺LKB1^fl/fl (n=3) mice were co-cultured with OP9-DL1 stromal cell monolayers. Cells were either stimulated with IL-7 or left untreated. Data show the mean fold increase in cell number following 5 days culture±SD. Differences in the proliferation of LckCre⁺LKB1^+/+ and LckCre⁺LKB1^fl/fl DN thymocytes are considered to be significant (p=0.007 without IL-7 and p=0.0005 in the presence of IL-7, respectively). The IL-7-induced proliferation of LckCre⁺LKB1^fl/fl DN thymocytes is also significant, p=0.004. (B) DN thymocytes from LckCre⁺LKB1^+/+ and CD44⁻CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice were co-cultured with OP9-DL1 stromal cell monolayers and treated with IL-7. The surface expression of CD4 and CD8 on thymocytes and the cell number were analyzed after 5 days of co-culture, representative of three independent experiments. C) DN4 thymocytes from LckCre⁺LKB1^+/+ and CD44⁻CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice were co-cultured with OP9-DL1. Flow cytometric analysis of forward and side scatter after 18 and 60 h of co-culture, respectively. (D) DN4 thymocytes from tamoxifen LckCre⁺LKB1^+/+ and CD44⁻ CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice were co-cultured with OP9-DL1 for 18 h, and stained with Thy1 and 7-aminoactinomycin D to investigate cell death. The numbers indicate the percentage of dead thymocytes. E) DN4 thymocytes from LckCre⁺LKB1^+/+ and CD44⁻ CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice were co-cultured with OP9-DL1 stromal cell monolayers. The data show the DNA content of DN4 thymocytes (not gated, total thymocyte population) after 18 h of co-culture. The numbers show the percentage of cells with degraded DNA.

Deletion of LKB1 prevents T-cell development (A) Appearance of thymi in LckCre⁺LKB1^fl/fl and LckCre⁺LKB1^+/+ mice. (B) Total thymocyte numbers from 5- to 8-wk-old LckCre⁺LKB1^fl/fl (n=10) and LckCre⁺LKB1^+/+ (n=10) mice. (C) Flow cytometry of CD4 and CD8 subpopulations in thymocytes from LckCre⁺LKB1^fl/fl and LckCre⁺LKB1^+/+ mice. Dot plots of Thy-1-gated cells are representative of ten independent experiments. (D) Flow cytometry of T cell (TCRβ⁺) and B cell (B220⁺) subpopulations in blood from LckCre⁺LKB1^fl/fl and LckCre⁺LKB1^+/+ mice. Dot plots are representative of three independent experiments. Gates show T and B cell populations. (E) Total numbers of T lymphocytes in spleens from LckCre⁺LKB1^fl/fl (n=5) and LckCre⁺LKB1^+/+ (n=5) mice. (F) Genomic PCR analysis of the fl/fl LKB1 gene in T lymphocytes from spleen indicating that peripheral T cells have not deleted the LKB1 allele.

PreTCR and Notch signaling in LKB1-null thymocytes (A) Intracellular TCRβ expression in DN3 and DN4 thymocytes of LckCre⁺LKB1^+/+ and DN3 and CD44⁻CD25^low thymocytes of LckCre⁺LKB1^fl/fl mice. (B) Flow cytometry of cellular DNA content (gated on live cells) of DN4 thymocyte from LckCre⁺LKB1^+/+ and CD44⁻CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice. The numbers indicate percentage of cells in S+G2/M phases of the cell cycle, representative of three independent experiments. (C) Data show CD98 and CD71 surface expression on DN4 thymocytes from LckCre⁺LKB1^+/+ and on CD44⁻CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice, representative of three independent experiments. (D) Histograms show Ser 235/236 S6 ribosomal protein phosphorylation in DN3 and DN4 thymocytes from LckCre⁺LKB1^+/+and in DN3 and CD44⁻CD25^low thymocytes from LckCre⁺LKB1^fl/fl mice, representative of three independent experiments. (E) Quantitative RT-PCR examining the expression levels of CD2, CD5, Nur77, Hes1 and Deltex1 mRNA in DN4 thymocytes of LckCre⁺LKB1^+/+ (n=3) and CD44⁻CD25^low thymocytes from LckCre⁺LKB1^fl/fl (n=5) mice. Expression was normalized to the mRNA levels in LKB^+/+ Lck-Cre⁺ DN4 thymocytes; data show mean±SD. Differences in CD2 and CD5 expression are considered to be significant (p=0.035 and p=0.036 for CD2 and CD5, respectively, p=0.1 for Nur77).

LKB1 is required for survival of proliferating peripheral T cells. (A) LKB1 Western blot of cell lysates from CreER^T2LKB1^fl/fl mouse T lymphoblasts either treated or not with 0.4 μM 4-hydroxytamoxifen for 3 days. (B) Fold increase in cell number of T lymphoblasts from CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl mice. The cells were treated with 0.4 μM 4-hydroxytamoxifen for 3 days prior to the start of analyses. Data show mean±SD. Differences in the proliferation of LckCre⁺LKB1^+/+ and LckCre⁺LKB1^fl/fl lymphoblasts are considered to be significant (p=0.001 at 24 h and p=0.0005 at 48 h). (C) Percentage of dead cells (based on FSC/SSC profile) in the 4-hydroxytamoxifen treated T lymphoblast cultures from CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl mice. The data corresponds to the 48 h time point in Fig. 5B. Data show mean±SD. Differences between LckCre⁺LKB1^+/+ and LckCre⁺LKB1^fl/fl samples are considered to be significant (p=0.003). (D) Primary T cells from CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl mice were either treated with IL-7 or stimulated with CD3/CD28 Ab-coated beads. The cells were treated with 0.6 μM 4-hydroxytamoxifen for 4 days prior to stimulation. Data show fold increase in cell number following 2 days of stimulation. Data show mean±SD. Differences between CD3/CD28 stimulated LckCre⁺LKB1^+/+ and LckCre⁺LKB1^fl/fl cells are considered to be significant (p=0.002). For IL-7 treated samples, p=0.15. (E) Naïve T cells from CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl mice were either treated with IL-7 or stimulated with CD3/CD28 Ab-coated beads. The cells were treated with 0.6 μM 4-hydroxytamoxifen for 4 days prior to the stimulation. Data shows the percentage of dead cells (based on FSC/SSC profile) following 2 days of stimulation. Data show mean±SD. Differences between CD3/CD28 stimulated LckCre⁺LKB1^+/+ and LckCre⁺LKB1^fl/fl cells are considered to be significant (p=0.00004). For IL-7 treated samples, p=0.3.