The authors have declared that no competing interests exist.
Conceived and designed the experiments: MZ JR DAC. Performed the experiments: MZ JR. Analyzed the data: MZ DAC. Wrote the paper: MZ DAC.
The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK). The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.
The adaptive immune response is mediated by T cells that express T cell antigen receptor complexes comprising of highly variable TCRα and β subunits
The balanced production of different T cell subpopulations, each with unique functions, during thymus development is essential to ensure the function and the homeostasis of the peripheral immune system. Hence, understanding the nature of the signals required for the development of different T cell subpopulations is important. All T cells that express αβ TCR complexes develop in the thymus from progenitors that lack expression of CD4 and CD8, hence termed double negative (DN) thymocytes. At the DN stage of thymocyte development T cell progenitors undergo genetic rearrangement of the TCRβ locus, which leads to the expression of a pre-TCR complex. This immature TCR complex drives DNs to proliferate and differentiate into CD4/8 double positive (DP) thymocytes. DP thymocytes that have successfully re-arranged their TCRα chain will undergo a selection process and differentiate to conventional TCR αβ CD4+ or CD8+ T cells, NKT cells or TCRαβ+ CD8αα+ gut lymphocytes.
In this context, there is currently considerable interest in understanding the signalling pathways that control metabolic checkpoints in T lymphocytes. It is thus relevant that recent studies have shown that the serine/threonine kinase LKB1 (Liver kinase B1 also known as serine/threonine kinase 11 - STK11) is important in controlling metabolic homeostasis in early T cell progenitors in the thymus
The studies to date about the role of AMPKα1 in T cells have thus been on the few mice that can compensate AMPKα1 loss in early embryo development. Accordingly, to directly compare the impact of AMPKα1 deletion and LKB1 deletion on thymus development there is a requirement to compare the consequences of selective deletion of either of these kinases at a defined stage of thymus development. We have therefore used a CD4Cre transgene to delete LKB1 or AMPKαl floxed alleles at the DP stage of thymocyte development. We found that LKB1 does not regulate survival of DP thymocytes although these cells fail to differentiate to conventional TCRα/β SP cell populations and are also defective in the development of iNKT cells and TCRα/β CD8αα IELs. In contrast, AMPKα1 null DPs produce normal numbers of both conventional and innate TCR αβ peripheral T cells. LKB1 is thus essential for the development of both conventional and innate TCR αβ T cells in the thymus but its mode of action is not through the activation of AMPK.
To explore the role of LKB1 in DP thymocytes, we backcrossed LKB1fl/fl mice to mice that express Cre recombinase under the control of the CD4 promoter. In this model,
(A) CD4pos thymocytes purified from LKB1fl/fl CD4Creneg or CD4Crepos thymi using MACS were lysed at 3×107 cells mL−1 in lysis buffer. Proteins extracted from cells and denatured were resolved on NuPAGE Bis-Tris 4–12% gels under reducing conditions and subsequent immunoblots were probed for indicated proteins, showing that LKB1 protein was efficiently deleted. GSK3 and β-catenin were used as loading controls for equal loading. Data are representative of two independent experiments. (B) Freshly isolated thymi from LKB1fl/fl CD4Creneg or LKB1fl/fl CD4Crepos mice were mashed to single cell suspensions. Cell number of double positive (DP) thymocytes was determined from a given volume using calibrated counting beads and from the frequency of cells co-stained for the MHC-receptors CD4 and CD8 of total number of thymocytes. Data are summary of four to six mice, where each symbol represents one mouse. (C) Single cell suspensions of freshly isolated thymi from LKB1fl/fl CD4Crepos or littermate controls were seeded at a cell density of 5−10×106 cells mL−1 in complete culture medium for 24 h. Frequency of live DP thymocytes was determined by staining for surface co-expression of CD4 and CD8 and exclusion of cells positive for the DNA binding dye DAPI. Statistical analysis using the Mann-Whitney test showed comparable frequencies of DAPIneg DP cells between LKB1fl/fl CD4Crepos and controls. Data summarise three independent experiments. (D) Thymocytes were isolated and 1×106 cells placed into the fibronectin-coated upper chamber of the transwell plate. Cells were left to migrate into the lower chamber containing medium only or 500 ng mL−1 CXCL12 for three hours. Cells from the lower chamber were collected and counted using counting beads using flow cytometry and the frequency of cells migrated was determined against the input that was used as putatively maximal migration capacity. Data summarise three independent experiments showing mean ± SEM.
LKB1fl/fl CD4Crepos mice had normal numbers of DP thymocytes but produced fewer TCRβhigh mature CD4 and CD8 SP thymocytes
LKB1fl/fl CD4Creneg and CD4Crepos thymi were isolated and analysed for (A) co-expression of CD4 and CD8 and (B) TCRβ expression. (B) Total number of TCRβhigh expressing thymocytes was quantified. (C) Ratio and total cell number of CD4pos and CD8pos TCRβhigh (SP) cells. (D) Analysis of lymphocyte populations in secondary lymphoid organs (spleen and lymph nodes). The frequency of B cells and T cells was determined by flow cytometric analysis for the expression of B220 and TCRβ, respectively. Quantification of total lymphocytes and TCRβpos lymphocytes is shown to the right showing that T cells were significantly reduced in secondary lymphoid tissues in LKB1fl/fl CD4Cre+ mice. Flow cytometric histograms and plots are representative of four experiments. Dot plots and bar graphs summarise data from at least four independent experiments. (E) Bi-parametric histogram shows frequency of TCRβpos and TCRγδpos T cells isolated from the epithelial layer of small intestines from LKB1fl/fl CD4Creneg and CD4Crepos mice followed by flow cytometric analysis. Data shown were gated on DAPIneg cells to identify live cells that remained intact following extraction and staining procedures. Dot plot summarises the frequencies of TCRβpos intraepithelial lymphocytes (IEL) from three mice per genotype. Statistical differences as indicated were determined using the Mann-Whitney test, where *p<0.05 and **p<0.01.
The transition of DPs to SPs can be staged by expression of the cell surface antigen CD69 and by the levels of TCR αβ complex expression
The analysis of CD69 and TCR levels on thymocytes from the LKB1fl/fl CD4Crepos mice shows that LKB1 null DP thymocytes respond to TCR triggering to up-regulate CD69 expression
(A) Thymi from LKB1fl/fl CD4Creneg and CD4Crepos mice were analysed for the co-expression of TCRβ and CD69 using flow cytometry as previously described
DP thymocytes also differentiate to produce CD4+ NKT cells that have an invariant Vα14 T cell receptor that recognises glycolipid antigens presented by the MHC-like molecule CD1d. In this context, there is evidence that there are different signalling requirements for the differentiation of iNKT cells and CD4 or CD8 SP mature T cells. For example, DP thymocytes lacking expression of Phospholipid-dependent kinase 1 (PDK1) fail to produce iNKT cells despite normal development of conventional α/β T cells
Thymocytes isolated from LKB1fl/fl CD4Crepos and littermate controls were stained for the presence of invariant NKT cells as described previously
LKB1 phosphorylates and activates AMPK
(A) CD4pos thymocytes from AMPKα1fl/fl CD4Creneg or CD4Crepos thymi were purified and lysed as described in
To explore more precisely the role of AMPK in thymocyte positive selection we backcrossed AMPKfl/fl CD4Crepos mice to mice expressing the defined OT1 αβ TCR transgene that select for class I restricted CD8 T cells. The data show that AMPK loss had no impact on the selection of thymocytes expressing the OT1 αβ TCR complex
(A) Blood biopsies were immune-phenotyped for the co-expression of the Vα2 TCR chain and CD8 and the frequency of Vα2pos CD8pos cells was determined in OT-1 TCRpos AMPKα1fl/fl CD4Creneg and CD4Crepos mice (n = 6). (B) Equal numbers of CD8pos OT-1 TCRpos AMPKfl/fl CD4Creneg and CD4Crepos lymphocytes were activated with 0.5 µM SIINFEKL peptide for 18 h. Supernatants were collected and subjected to ELISA for the detection of soluble IFNγ secreted by activated T cells. Data summarise mean amounts of IFNγ of three mice analysed in technical triplicates. Statistical differences as indicated were determined using Mann-Whitney test.
Previous studies have shown that LKB1 controls the survival of T cell progenitors at the DN stage of development. In these studies LKB1 was deleted at the DN2/3 stage of thymocyte development using the LckCre recombinase model. In LckCre LKB1fl/fl mice a few DP thymocytes survive the early deletion of LKB1 and become DP thymocytes
The role for LKB1 in the development of conventional αβ TCR T cells has been previously reported
It has been proposed that LKB1 controls thymus development via its substrate AMPK
In this respect it is important to note that LKB1 phosphorylates and activates multiple members of the AMP-activated protein kinase (AMPK) subfamily such as the salt inducible kinase, the Par/MARK kinases and NUAK1
Mice as outlined next were bred and maintained under specific pathogen-free conditions in the Biological Resource Unit at the University of Dundee. The procedures used were approved by the University Ethical Review Committee, a committee of the University Court, at its meeting on 19th December 2007 and then authorised by a project licence under the UK Home Office Animals (Scientific Procedures) Act 1986 issued by the Home Office on 14th April 2008.
LKB1fl/fl mice were generated and bred as previously described by Sakamoto
Single cell suspensions of freshly isolated thymi were maintained at 5−10×106 cells mL−1 in DMEM containing 10% heat-inactivated foetal calf serum (FCS) (Life Technologies, UK), 50 µM 2-mercaptoethanol (Sigma-Aldrich, Germany), 100 U mL−1 penicillin and 100 µg mL−1 streptomycin (Life Technologies, UK). Lymph nodes and spleens were gently disaggregated. Disaggregated spleens were also treated for lysis of red blood cells. Lymph nodes and spleens were re-suspended at 5−10×106 cells mL−1 in RPMI-1640 containing L-glutamine and supplemented with 10% heat-inactivated FCS, 50 µM 2-mercaptoethanol, 100 U mL−1 penicillin and 100 µg mL−1 streptomycin. Primary CD8pos T cells (4×106 cells mL−1) from OT1-TCR transgenic mice were activated with 0.5 µM soluble ovalbumin-derived SIINFEKL peptide for 18 h in 96-well plates and supernatants were collected followed by cytokine secretion assays. IFNγ secretion was determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit from eBiosciences.
Thymocytes (1×108 cells) were labelled with biotinylated anti-CD4 (BD Pharmingen) and CD4pos thymocytes were isolated using streptavidin-coated magnetic beads by autoMACS (Miltenyi Biotec, Germany). The positive fraction collected was then lysed for immunoblotting.
Thymocytes (3×107 cells) were lysed in 1 mL of F buffer [10 mM Tris-HCl pH 7.05, 50 mM NaCl, 30 mM Na-pyrophosphate, 50 mM NaF, 5 µM ZnCl2, 10% Glycerol, 1% NP-40, 1 mM DTT] supplemented with 50 nM calyculin A for 15 min on ice and centrifuged for 20 min at 1.32×104 rpm. Lysates were mixed and boiled with NuPAGE LDS sample buffer (Life Technologies) supplemented with 100 mM DTT. Samples were separated on NuPAGE Bis-Tris 4–12% gradient gels (Life Technologies) at 200 V for up to 60 min under reducing conditions. Separated proteins were transferred onto Hybond™-C Super nitrocellulose membrane (Amersham Biosciences, UK) at 30 V for 150 min in Novex XCell II Modules (Invitrogen, UK) at 4°C. Membranes were blocked with 5% dry milk/PBS supplemented with 0.5% Tween-20 (Sigma) and probed for indicated pan-proteins. Anti-AMPKα1 was a kind gift of Grahame Hardie, University of Dundee. Anti-Smc1 was obtained from Bethyl Laboratories Inc. All other antibodies for immunoblotting were obtained from Cell Signaling Technology.
Freshly isolated small intestines were freed from mesenteric lymph nodes, Peyer's patches, debris, adipose and connective tissues. Digested food was removed mechanically and the intestinal lumen was cleaned using PBS. Intestines were opened longitudinally and then cut into 1-cm-pieces, which were incubated in Ca2+/Mg2+-free PBS (Sigma) supplemented with 10% filtered heat-inactivated FCS, 1 mM Na pyruvate, 20 mM HEPES pH 8.0, 10 mM EDTA pH 8.0 and 10 µg mL−1 Polymyxin B for 30 min at 230 rpm and 37 °C. Tissue suspensions were filtered using a 70 µm-filter cell strainer (BD Falcon) and cells were collected by centrifugation. Cells were re-suspended in 37.5% isotonic percoll (Sigma) and collected by centrifugation (without break). Following careful recovery of the cell pellet, cells were washed, re-suspended in complete RPMI-1640 culture medium, filtered through a 40- µm-filter (BD Falcon) and stained for flow cytometric analysis.
Accurate cell counts of lymphocyte cultures were taken by using AccuCheck counting beads (Life Technologies, UK). One to two million cells of freshly disaggregated secondary lymphoid organs or extracted from small intestines were incubated with FC Block (BD Pharmingen) for 10 min at 4°C in RPMI-1640 or PBS supplemented with 1% FCS (FACS buffer). FC Block was omitted for thymocyte suspensions. Cells were labelled with saturating concentrations of antibody in FACS buffer. Antibodies used were conjugated to fluorescein-isothiocyanate, phycoerythrin (PE), peridinin-chlorophyll protein (PerCP)-Cy.5.5, PE-Cy7, allophycocyanin (APC), APC-Cy7 or –eFluor®780, Horizon V450 or V500, Alexa Fluor®700 as obtained from BD Pharmingen or eBiosciences: anti-CD4 (L3T4), anti-CD8α (53-6.7), anti-CD8β (H35-17.2), anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-Vα2 TCR (B20.1), anti-Vβ5.1/5.2 TCR (MR9-4), anti-TCRβ (H57-597), anti-TCRγδ (GL3) and anti-CD24 (M1/69). Staining for Vα14 TCR to detect NKT cells was performed as described previously
Migration assays were performed using Transwell chemotaxis plates (CoStar). Membrane inserts of transwell plates were coated with 5 µg mL−1 fibronectin at 4 °C over night. Membranes were then blocked with 2% heat-inactivated FCS/PBS for one hour at 37 °C. Freshly isolated thymocytes (1×106 cells in 100 µL of complete DMEM medium) were placed in the upper chamber of the transwell plate in triplicate. Culture medium without or with CXCL12 (500 ng mL−1 in 600 µL) was placed in the lower chamber. After 3 h of incubation at 37 °C in 5% CO2, the percentage of cells against the input control was determined using flow cytometry.
Quantified data were evaluated using non-parametric Mann-Whitney test, where experimental numbers were not sufficient to prove normal distribution. Bar graphs are shown as mean ± standard deviation unless otherwise stated. GraphPad Prism 4.0c or later for Mac OS X was used for statistical evaluation and generation of bar graphs and dot plots of quantified data.
We would specifically like to thank Arlene Whigham for her assistance in preparing and performing the flow cytometric analysis of gut lymphocytes. We would like to thank the Cantrell lab in the College of Life Sciences, University of Dundee, for critical comments and reading of the manuscript. We thank Elizabeth Emslie for her help, Grahame Hardie and Benoit Viollet for providing reagents and AMPKfl/fl mice, respectively. We would like to thank the Wellcome Trust Flow Cytometry Facility of the University of Dundee for technical support and advice, and the Biological Resource Unit for the animal care.