LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells

Deletion of LKB1 prevents T-cell development

The objective of the present study was to explore the role of LKB1 in T lymphocytes. Mice with floxed LKB1 exons 4–7 on both alleles (LKB1^fl/fl) ³⁴ were backcrossed with mice expressing Cre recombinase under the control of the proximal p56lck proximal promoter (Lck-Cre⁺), which induces Cre expression in T-cell progenitors in the thymus ³⁵. The LckCre⁺LKB1^fl/fl mice were viable, although they had very small thymi (Fig. 1A) that lacked the normal cortical/medullar architecture (data not shown). These small LckCre⁺ LKB1^fl/fl thymi contained greatly reduced numbers of thymocytes compared to LckCre⁺LKB1^+/+ controls (Fig. 1B). To determine which stages of thymocyte development were sensitive to LKB1 loss, LckCre⁺LKB1^fl/fl thymi were analyzed for expression of the major histocompatibility complex (MHC) receptors CD4 and CD8. Early T-cell progenitors are double negative (DN) for CD4 and CD8. DN progenitors undergo TCR-β locus rearrangements to produce a TCR-β polypeptide that permits surface expression of the pre-TCR complex. The pre-TCR then supports survival and directs rapid clonal expansion along with differentiation of cells into CD4⁺CD8⁺ double positive (DP) thymocytes. TCR α-chain gene rearrangements then occur and cells that express a functional but non self reactive α/β TCR complex differentiate to either CD4⁺ or CD8⁺ single positive (SP) T cells ^1,3,36. Figure 1C shows that LckCre⁺LKB1^fl/fl thymi contained mostly DN cells and had very few DP or SP. There were also very few peripheral T cells present in the blood or spleen of the LckCre⁺LKB1^fl/fl mice compared to LckCre⁺ LKB1^+/+ control animals (Fig. 1D and E). Moreover, in the few peripheral T cells of LckCre⁺LKB1^fl/fl mice there was evidence that LKB1 deletion was not complete, as cells contained non-deleted LKB1 floxed alleles (Fig. 1F). Initial analysis of LckCre⁺LKB1^fl/fl mice thus showed that LKB1 is essential for T-cell development in the thymus; only cells that fail to delete LKB1 can differentiate in the thymus and generate peripheral T cells.

DN3/DN4 transition defects in LKB1-null thymocytes

DN thymocytes can be subdivided on the basis of CD44 and CD25 expression: the earliest progenitors are CD44⁺and CD25⁻ DN1), followed sequentially by the CD44⁺ and CD25⁺ (DN2), CD44⁻ and CD25⁺ (DN3) and CD44⁻CD25⁻ DN4 ) populations ^3,36. CD25 and CD44 staining profiles showed that LckCre⁺LKB1^fl/fl thymi had an increased frequency of the DN3 subset: numbers of DN3 thymocytes were 4.0–9.1×10⁶ in LckCre⁺LKB1^+/+ while 3.6–7.6×10⁶ in LckCre⁺LKB1^fl/fl thymi (Fig. 2A). Moreover, DN3 cells in LckCre⁺LKB1^fl/fl thymi showed abnormally high levels of CD25 expression (Fig. 2B). There was no subset of cells corresponding to WT DN4, i.e. CD44⁻CD25⁻ in LckCre⁺ LKB1^fl/fl thymi although there was a population of CD44⁻ CD25^low cells that resembled cells in transition from the DN3 to DN4 stage. Numbers of DN4 thymocytes were 1.6–3.7×10⁶ in LckCre⁺LKB1^+/+ thymi while the numbers of CD44⁻CD25^low thymocytes were 3.6–7.6 and 0.8–2.1×10⁶ in LckCre⁺LKB1^fl/fl samples. The first phenotypic difference between LckCre⁺LKB1^fl/fl and normal thymi is thus seen in DN3 thymocytes; some cells partially transit to the DN4 stage albeit at lower frequency than normal. The proximal p56Lck promoter is active in DN1 cells, although its activation tends to be heterogeneous until the DN3 stage ³⁷. In this context, genomic PCR analysis indicated partial deletion of LKB1 in DN3 cells and complete loss in the CD44⁻ CD25^low cells (Fig. 2C).

LKB1 is known to phosphorylate and activate AMPK in response to energy stress (22,38. Therefore to assess functional loss of LKB1, we compared the ability of 2-deoxyglucose, an inhibitor of glycolysis that increases cellular ratios of AMP/ATP, to activate AMPK in control and LckCre⁺LKB1^fl/fl DN thymocytes. AMPKα1 can also be phosphorylated and activated by calcium/calmodulin dependent kinase kinases (CaMKK) in T cells ³¹ so the effect of the CaMKK inhibitor STO609 on AMPK activation was also examined. Figure 2D shows that there is low basal phosphorylation of Thr 172 in the activation loop of AMPKα1 in WT pre-T cells and that this phosphorylation is increased by 2-DG treatment. AMPKα1 Thr 172 phosphorylation was not inhibited by STO609. Basal phosphorylation of AMPKα1 in LckCre⁺LKB1^fl/fl thymocytes was observed, but this was not increased by 2-DG treatment.

LKB1 and pre-TCR/Notch signaling

CD25 downregulation in DN3 is driven by the pre-TCR ³⁹. The high level of CD25 on LckCre⁺LKB1^fl/fl DN3 cells coupled with the failure of these cells to completely downregulate CD25 and complete transit to DN4 cells could thus reflect a problem with pre-TCR expression or function. The rate-limiting step for pre-TCR expression is normally the TCR β locus rearrangement ⁴⁰. However, analysis of intracellular TCR-β expression showed that LckCre⁺LKB1^fl/fl DN3 cells had successfully rearranged their TCR-β locus and expressed intracellular TCR subunits at the normal frequency (approximately 15%) (Fig. 3A). It was also seen that the subpopulation of LKB1-null pre-T cells that had partially downregulated CD25 were predominantly TCR-β selected (80%) and in this regard were indistinguishable from bona fide WT DN4 (Fig. 3A).

One difference between WT and LKB1 deleted DN3 cells is that the latter have increased expression of CD25, a phenotype that frequently results from defective pre-TCR signal transduction. We therefore examined a range of pre-TCR-induced responses in LckCre⁺LKB1^fl/fl pre-T cells. One key function of the pre-TCR is to drive cell cycle progression as cells transit from DN3 to DN4. Flow cytometric analysis of cellular DNA content showed that LKB1 loss did not block cell cycle entry, as LKB1-null pre-T cells can enter the proliferative S/G2 phases of the cell cycle albeit at a reduced frequency compared to controls (Fig. 3B). Pre-TCR signaling also induces expression of key nutrient receptors on T-cell progenitors, namely CD71 the transferrin receptor and CD98, a subunit of the l-aa transporter ⁷. LKB1 loss had no impact on surface expression of CD71 the transferrin receptor but there was a striking decrease in the surface expression of CD98 aa transporter in LckCre⁺LKB1^fl/f pre-T cells (Fig. 3C).

We also analyzed the effect of LKB1 deletion on pre-TCR-induced phosphorylation (Ser235/236) of the S6 ribosomal subunit by the 70 kDa ribosomal S6 kinases. S6 phosphorylation can be quantified directly ex vivo by flow cytometry and intracellular staining of fixed cells with specific phospho-S6 antisera ¹⁰. WT DN3 thymocytes analyzed immediately ex vivo are heterogeneous for phosphoS6: the majority of cells are phosphoS6^low but 15–20% are phosphoS6^high corresponding to cells that have rearranged their TCR-β subunit to express the pre-TCR. DN4 thymocytes are primarily phosphoS6^high (Fig. 3D). The data show severely reduced phosphorylation of S6 in LckCre⁺LKB1^fl/fl stained DN thymocytes. S6 phosphorylation is induced by PDK1/PKB signaling pathways initiated by the pre-TCR and sustained by stromal signals such as Notch ligands ⁴¹. In this respect, LKB1-null T cells had normal expression of CD71, the transferrin receptor. This is relevant because CD71 expression in T-cell progenitors is controlled by pre-TCR/Notch-induced PDK1/PKB-mediated pathways ⁷. The normal expression of CD71 is thus evidence for functioning PKB signaling in LKB1-null pre-T cells. We further explored the impact of LKB1 on pre-TCR signaling by examining expression of known pre-TCR-induced genes, such as CD2 and CD5 and Nurr77 ^7,42,43. LKB1-null DN4 pre-T cells expressed lower levels of CD2 mRNA than controls and there was also a small but reproducible decrease in expression of CD5 mRNA. However, the expression of Nur77, also a pre-TCR-induced gene, was not changed (Fig. 3E). The results argue that LKB1 loss causes selective defects but not global problems with pre-TCR signal transduction. We also examined the expression of Notch target genes, Hes-1 and Deltex-1⁴⁴, in pre-T cells isolated ex vivo from control or LckCre⁺LKB1^fl/fl mice. The CD44⁻CD25^low cells from LckCre⁺LKB1^fl/fl mice expressed increased levels of Hes-1 and Deltex1 when compared with control cells (Fig. 3E). The expression of these Notch target genes in LKB1-null pre-T cells argues that these cells are responding to Notch ligands in vivo.

LKB1 is required for survival of TCR-β selected T-cell progenitors

The differentiation and proliferation of β selected pre-T cells in the thymus is dependent on sustained Notch receptor/ligand interactions ^8,41,45. To explore further impact of LKB1 deletion on thymocyte development, we compared the responses of pre-T cells from control or LckCre⁺LKB1^fl/fl mice in an in vitro system that uses OP9 stromal cells expressing the Notch ligand delta-like 1 (OP9-DL1) and IL-7 to support survival, proliferation and differentiation of T-cell progenitors ⁴⁶. In initial experiments we compared the responses of cells maintained on the OP9-DL1 cells in both the presence and absence of IL-7. As described previously, normal DN thymocytes (LckCre⁺LKB1^+/+) undergo proliferative expansion when cultured on OP9-DL1 cells and this proliferative response is enhanced by the addition of IL-7 ⁴⁷ (Fig. 4A). In contrast, LKB1-null DN thymocytes (LckCre⁺LKB1^fl/fl) did not proliferate on OP9-DL1 cells. The addition of IL-7 to the cultures caused a small improvement to the proliferative response of the LKB1-null DN thymocytes, indicating that the cells had not lost the capacity to respond to IL-7 but the cells still failed to undergo proliferative expansion in these in vitro models. Moreover, the proliferative expansion of normal TCR-β selected DN4 (LckCre⁺LKB1^+/+) is followed by their differentiation to DP thymocytes. Strikingly, TCR-β selected CD44⁻CD25^lowLckCre⁺LKB1^fl/fl thymocytes do not differentiate to DP on OP9-DL1 cells (Fig. 4B), in fact they die. LKB1-null pre-T cells thus rapidly acquire the light scattering profiles of dead cells rather than viable cells (Fig. 4C). LKB1-null pre-T cells also show a rapid decrease in plasma membrane integrity (Fig. 4D) and a high frequency of cells with a sub-G1 DNA content, indicative of DNA degradation (Fig. 4E). LKB1 is thus essential for the survival of TCR-β selected T-cell progenitors as they proliferate and differentiate in response to Notch ligands and IL-7.

LKB1 is required for survival of proliferating peripheral T cells

Does this requirement for LKB1 support the proliferation of T-cell progenitors unique to this T-cell subpopulation? To address this question we examined the LKB1 requirement for the proliferative responses of mature T cells. In these experiments we bypassed the LKB1 requirement for T-cell development by breeding mice expressing LKB1 floxed alleles to CreER^T2 mice that express a tamoxifen inducible Cre recombinase. In initial experiments, CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl peripheral T cells were polyclonally activated with CD3 Ab and maintained in exponential proliferation in vitro by the addition of IL-2. T cells were then treated with 4-hydroxytamoxifen (4OHT) to induce deletion of LKB1 in the CreER^T2LKB1^fl/fl cells. The data (Fig. 5A) confirmed deletion of LKB1 following 4OHT treatment of CreER^T2LKB1^fl/fl peripheral T lymphoblasts and revealed that LKB1 deletion stopped the proliferation of T cells and resulted in cell death (Fig. 5B and C). In further experiments, peripheral primary T cell isolated from CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl mice were cultured in IL-7 in the presence of 4OHT. Thereafter cells were stimulated with CD3/CD28 Ab coated beads to induce proliferation. The data in Fig. 5D show that 4OHT treated control lymphocytes from CreER^T2 mice (CreER^T2LKB1^+/+) gave a robust proliferative response following CD3/CD28 stimulation whereas CreER^T2LKB1^fl/fl T cells did not. Both cell populations could be maintained with comparable viability when cultured in IL-7 (Fig. 5E) but the viability of the CD3/CD38 triggered LKB1-null cells were markedly reduced compared with the control LKB1 WT cells (Fig. 5E). These data show that LKB1 is required to support the survival of mature T cells when they are induced to proliferate.

Discussion

The present results establish that LKB1 has critical functions during T-cell development. In the thymus LKB1 is required for the survival and differentiation of TCR-β selected T-cell progenitors. T-cell progenitors that successfully rearrange their TCR-β and express the pre-TCR complex normally proliferate rapidly prior to differentiation to the DP stage of thymus development. The present data now show that LKB1-null pre-T cells that have rearranged their TCR-β locus are unable to proliferate and undergo cell death rather than complete their normal program of development. The data also show that LKB1-null peripheral T cells are unable to proliferate and undergo cell death in response to CD3/CD28 stimulation. Activated T cells maintained in culture with IL-2 similarly fail to proliferate and die following LKB1 loss. It is well established that cells that fail to match energy production to energy demands die by apoptosis. LKB1 thus appears to coordinate the ability of T cells to match energy production to the energy requirements for the proliferative burst that accompanies TCR-β selection or the immune activation/cytokine-induced proliferation of peripheral T cells.

LKB1 phosphorylates and activates multiple members of the AMPK family including the α1 and α2 isoforms of AMPK, NUAK1-2, BRSK1-2, QIK, QSK, SIK, MELK and MARK1–4 kinases ²¹. There have been some studies to probe the role of individual members of this kinase family in T cells. For example, AMPK α1-null T cells have increased sensitivity to energy stress ³² and there are defects in immune homeostasis in mice that lack expression of another AMPK family kinase, MARK2/Par-1b ³³. However, neither the AMPK α1 nor MARK2 mice showed any evidence for defective thymus development ^32,33. This could mean that there is redundancy between members of the AMPK family in T cells. In this respect, redundancy between different serine/threonine kinases has been seen previously in T lymphocytes. For example, T cells express three isoforms of PKB/Akt and unveiling of the function of these kinases in vivo in the thymus required simultaneous deletion of all three isoforms ⁴⁸. There is also redundancy between different isoforms of PI3K in T-cell progenitors such that deletion of both the p110 delta and p110 gamma catalytic subunits of PI3K is required to reveal the importance of PI3K in T-cell progenitors ⁴⁹. The impact of LKB1 deletion versus deletion of individual AMPK family kinases is an indication that there is similar redundancy between different members of the AMPK family in T cells.

Previous studies have focused extensively on the role of PI3K and the serine/threonine kinases PKB/Akt in the control of T-cell metabolism ⁵⁰.The present data showing that LKB1, a kinase that evolved to couple energy metabolism and cell differentiation, also has essential functions in T lymphocytes. It reveals that T cells use multiple pathways to deal with the metabolic stress of proliferation. Here it is important to note that these two kinase pathways function in quite different modes. In T cells, PKB/Akt activity is controlled directly by Ag receptors/costimulatory molecules and or cytokines and the level of Akt activity is determined by cellular levels of Phosphatidyinositol (3,4,5) tris phosphate and the activity of PDK1 and mTOR (mammalian target of rapamycin) ⁵¹. In contrast, LKB1 signaling pathways are not coupled directly to extracellular stimuli but rather act homeostatically in response to energy stress to restore energy balance. For example, LKB1 acts as a rheostat for cellular AMP/ATP ratios and phsophorylates and activates AMPK when cellular levels of ATP drop. This concept of how LKB1 works explains why it is not possible to explain the phenotype of LKB1-null T cells by the loss of a linear biochemical signaling pathway that is coupled to a single receptor. Rather LKB1 is integral to the control of T-cell proliferation irrespective of the proliferative stimulus.

Mice

Mice (5–7 weeks old) were maintained in SPF conditions at the University of Dundee under Home Office project license PPL60/3116. C57BL/6-Gt(ROSA)26Sor tm9(cre/Esr1)Arte mice carrying a transgene in the ROSA locus encoding for a ubiquitously expressed Cre recombinase/modified oestrogen receptor fusion protein CreER^T2, were purchased from TaconicArtemis. LKB1^fl/fl mice were generated and bred as previously described ³⁴. LKB1^fl/fl mice were crossed either to transgenic mice expressing Cre recombinase under the T-cell-specific Lck promoter ³⁵ or to CreER^T2 mice. Genotyping was performed by PCR using genomic DNA isolated from ears. The presence of a WT or floxed LKB1 allele was detected using two primers: 5′-CCAGCCTTCTGACTCTCAGG-3′ and 5′-GTAGGTATTCCAGGCCGTCA-3′. PCR amplification of the WT or the floxed LKB1 alleles resulted in 200-bp and 250-bp PCR products, respectively. For the detection of Cre (either LckCre or CreER^T2), the following primers were employed: 5′-AAATGGTTTCCCGCAGAACC-3′ and 5′-TAGCTGGCTGGTGGCAGATG-3′. Cre-mediated deletion of the floxed LKB1 allele was proved by PCR using primers: 5′-CAGGTTCAGCAGAATCAC-3′ and 5′-GGGAAGAAGGCGATATAAGG-3′.

Flow cytometric analysis

Characterization of different thymocyte subpopulations was performed as described previously ⁷. Briefly, Ab conjugated to fluorescein isothiocyanate, phycoerythrin, allophycocyanin and biotin were obtained from either Pharmingen (San Diego, CA, USA) or eBioscience (San Diego, CA, USA). TriColour-conjugated Ab were obtained from Caltag (Burlingame, CA, USA). Cells were stained for surface expression of the following markers using the Ab in parentheses: CD4 (RM4-5), CD8 (53-6.7), CD25 (7D4), CD44 (IM7), CD98 (RL388), CD71 (C2), Thy1.2 (53-2.1), TCR-β (H57-597), B220 (RA3-6B2) and TCR/ (GL3). Cells were stained with saturating concentrations of Ab in accordance with the manufacturer's instructions. Data were acquired on either a FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA) or a LSR1 flow cytometer (Becton Dickinson) using CellQuest software and were analyzed using either CellQuest (Becton Dickinson) or FlowJo (Treestar, San Carlos, CA, USA) software. Viable cells were gated according to their forward scatter and side scatter profiles. CD4⁻ and CD8⁻ DN subsets were gated by lineage exclusion (lineage) of all CD4⁺, CD8⁺ DP and SP cells and TCR-γ⁺. DN3 and DN4 were further defined as CD25⁺CD44⁻ and CD25⁻CD44⁻ thymocytes, respectively. Mature SP thymocytes were defined as Thy-1⁺, TCR-β^hi and positive for either CD4 or CD8 expression. Cell death was analyzed by staining with 7-aminoactinomycin D (5 μg/mL).

Cell Purification

DN3 and DN4 thymocytes were purified by first depleting thymic populations of CD4⁺ and CD8⁺ cells using an AutoMACs magnetic cell sorter (Miltenyi Biotech, Auburn, CA, USA) before sorting to a purity greater than 95%, using a FACS VantageSE cell sorter (Becton Dickinson). Peripheral T lymphocyes (from spleen) were purified by AutoMACS magnetic cell sorter (Miltenyi Biotech) using mouse pan T-cell isolation kit.

Intracellular TCR-β staining

Intracellular TCR-β staining was performed as described previously ¹⁰. Briefly: thymocytes were stained for cell surface markers to define DN3 and DN4 subsets. After fixation in 1% paraformaldehyde for 10 min at 25°C, cells were washed in PBS and permeabilized for 10 min at room temperature in saponin buffer (0.5% (weight/volume) saponin, 5% FBS and 10 mM HEPES, pH 7.4, in PBS) Permeabilized cells were incubated for 45 min with phycoerythrin-conjugated Ab to TCR in saponin buffer, were washed in saponin buffer and were analyzed on a FACS Calibur. Cell surface binding sites were blocked by biotinylated TCR and the specificity of staining was controlled by parallel staining with phycoerythrin-conjugated isotype-matched control Ab (Armenian hamster IgG2).

Intracellular phospho-S6 staining

Intracellular phosphoS6 staining was performed as described previously ¹⁰. Thymocytes were treated with 20 nM rapamycin or were left untreated for 20 min at 37°C. Treatment of cells with rapamycin inhibits the activity of mTor and rapidly reverses S6 (Ser 235/236) phosphorylation. Phospho-S6 staining of rapamycin-treated cells thus provides an internal negative control as a standard for each sample. Cells were washed and stained with surface markers to define the DN3 and DN4 subsets, then were fixed in 0.5% paraformaldehyde for 15 min at 37°C, followed by 15 min in 90% methanol on ice. After fixation, cells were washed twice in BSA buffer (0.5% BSA in PBS), then blocked for 10 min at 25°C in BSA buffer. Cells were incubated for 30 min at 25°C with Ab to phospho-S6 (2211; Cell Signaling Technologies) in BSA buffer, then were washed and incubated for 30 min at 25°C with FITC-conjugated donkey IgG Ab to rabbit (Jackson ImmunoResearch). Samples were washed in BSA buffer and were analyzed on a FACS Calibur.

Cell cycle analysis

The cellular DNA content of DN3 and DN4 thymocytes was analyzed on live cells, with Hoechst-33342 (Molecular Probes) staining. Cells were incubated for 1 h at 37°C in 5 μg/mL Hoechst-33342 in 2% FBS DMEM and then were surface stained for CD25, Thy-1 and lineage markers (including CD44). Samples were analyzed on an LSR1 flow cytometer eliminating doublets from the analysis.

Quantitative RT-PCR

DN3 and DN4 Cells (0.5×10⁶ per sample) were lysed for RNA isolation using the QIAshredder homogenizer (QIAGEN). RNA was purified with the RNeasy Mini, RNA isolation kit (QIAGEN) following the manufacturer's protocol and including an on-column DNase digestion step with the RNase-free DNase set (QIAGEN). cDNA was generated using the iScript™ cDNA synthesis kit (BIO-RAD). Real-time PCR reactions were performed in a 20 μL mixture containing 1× iQ™ SYBR Green supermix (BIO-RAD), 1 μL of cDNA preparation and 0.4 μM forward and reverse primers. Real-time quantitations were performed using the BIO-RAD iCycler iQ system and using 18S rRNA as an internal control. The primer sequences for the PCR were as follows

Cell culture

OP9 bone marrow stromal cells expressing OP9-DL1 and control OP9 cells were a gift from Juan Carlos Zúñiga-Pflücker (Toronto, Canada) ⁴⁶. OP9 cells were maintained in αMEM supplemented with 50 μM 2-mercaptoethanol, 100 U/mL penicillin, 1 mg/mL streptomycin and 20% heat-inactivated FBS. DN thymocytes were co-cultured on OP9-DL1 monolayers for times indicated in figure legends in the presence or absence of 5 ng/mL of IL-7 as indicated. For harvesting, thymocytes were filtered through 50 μm filters to remove OP9-DL1 cells before developmental progression of T lineage cells was assessed. For activation of primary T cells spleens or lymph nodes were removed from CreER^T2LKB1^+/+ and CreER^T2LKB1^fl/fl mice, disaggregated and red blood cells were lysed. Cells were cultured in RPMI-1640 medium containing l-glutamine (Invitrogen), 10% v/v heat-inactivated FBS (Gibco), 50 μM β-mercaptoethanol (Sigma) and penicillin–streptomycin (Gibco).

Single-cell suspensions from lymph node preparations cultured at 5×10⁶ cells per mL. Cells were treated with 0.6 μM 4OHT in the presence of 5 ng/mL IL-7 for 4 days. Thereafter cells were either stimulated with CD3/CD28 Ab-coated beads (Dynabeads Mouse CD3/CD28T cell expander, Invitrogen) or left in 5 ng/mL IL-7 for 3 days.

For the generation of lymphoblasts, mouse CD8⁺ T cells were cultured for 48 h in the presence of a CD3 Ab (5 μg/mL; 145-2C11; R&D Systems) to trigger the TCR. Thereafter the cells were washed and maintained in exponential proliferation with 20 ng/mL IL-2 (Chiron) for another 2 days. Then the cells were treated with 0.4 μM 4OHT for 3 days. The 4OHT was washed and the cell proliferation was investigated in the presence of IL-2.

Western blot analysis

Sorted DN4 thymocytes or peripheral T lymphoblasts were lysed on ice in NP-40 lysis buffer (50 mM HEPES (pH 7.4), 75 mM NaCl, 1% Nonidet P-40, 10 mM sodium fluoride, 10 mM iodoacetamide, 1 mM EDTA, 40 mM β-glycerophosphate, protease inhibitors, 100 μM sodium orthovanadate). Lysates were centrifuged at 1600×g for 15 min at 4°C. Protein samples were separated by SDS NuPAGE 4–12% Bis-Tris PAGE (Invitrogen) transferred to nitrocellulose membrane and detected by Western blot analysis using standard techniques. The phosphorylated AMPK (phospho-172Thr-AMPK Rabbit mAb (Cell Signaling Technology) and α1 AMPK antisera were used as described ³¹. The LKB1 Ab from Cell Signaling Technology was used to probe for LKB1 expression.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 4.00 for Macintosh, GraphPad Software. A non-parametric Mann–Whitney test was used where the number of experiments performed was not sufficient to prove normal distribution. When comparing gene expression between LckCre⁺LKB1^+/+ and LckCre⁺LKB1^fl/fl animals, a Student's t-test was used with the theoretical mean of LckCre⁺LKB1^+/+ sample set to 1. Differences were considered as significant if p<0.05.