Adenosine-mono-phosphate-activated protein kinase-independent effects of metformin in T cells

The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK

The role of ezrin/radixin/moesin(ERM) proteins in L-selectin-dependent signalling

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Double positive thymocytes survive without LKB1.

<p>(A) CD4^pos thymocytes purified from LKB1^fl/fl CD4Cre^neg or CD4Cre^pos 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 LKB1^fl/fl CD4Cre^neg or LKB1^fl/fl CD4Cre^pos 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 LKB1^fl/fl CD4Cre^pos 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 DAPI^neg DP cells between LKB1^fl/fl CD4Cre^pos 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.</p

LKB1 is required for the progression of positive selection and maturation of T cells.

<p>(A) Thymi from LKB1^fl/fl CD4Cre^neg and CD4Cre^pos mice were analysed for the co-expression of TCRβ and CD69 using flow cytometry as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone.0060217-Tamas1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone.0060217-Cao1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone.0060217-Lesourne1" target="_blank">[13]</a>. (B) Flow cytometric analysis of TCRβ^high cells stained for the surface expression of CD24 (heat stable antigen) and CD62L (L-selectin). Data shown are representative of at least three mice.</p

LKB1 required for accumulation of T cells in thymus, lymphoid organs and the small intestine.

<p>LKB1^fl/fl CD4Cre^neg and CD4Cre^pos thymi were isolated and analysed for (A) co-expression of CD4 and CD8 and (B) TCRβ expression. (B) Total number of TCRβ^high expressing thymocytes was quantified. (C) Ratio and total cell number of CD4^pos and CD8^pos TCRβ^high (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 TCRβ^pos lymphocytes is shown to the right showing that T cells were significantly reduced in secondary lymphoid tissues in LKB1^fl/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 TCRβ^pos and TCRγδ^pos T cells isolated from the epithelial layer of small intestines from LKB1^fl/fl CD4Cre^neg and CD4Cre^pos mice followed by flow cytometric analysis. Data shown were gated on DAPI^neg cells to identify live cells that remained intact following extraction and staining procedures. Dot plot summarises the frequencies of TCRβ^pos 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.</p

LKB1 is required for the accumulation of thymic iNKT cells.

<p>Thymocytes isolated from LKB1^fl/fl CD4Cre^pos and littermate controls were stained for the presence of invariant NKT cells as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone.0060217-Tamas1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone.0060217-Finlay1" target="_blank">[14]</a>. iNKT cells were identified as CD24^low CD1d-αGalCer^pos thymocyte population. Flow cytometric histograms and frequencies shown are representative of at least three independent experiments. Bar graph summarises total number of CD24^low CD1d-αGalCer^pos thymocytes from three mice. Statistical difference between groups as shown was determined using Mann-Whitney test where * p<0.05.</p

Development and accumulation of T cells does not require AMPKα1.

<p>(A) CD4^pos thymocytes from AMPKα1^fl/fl CD4Cre^neg or CD4Cre^pos thymi were purified and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone-0060217-g001" target="_blank">Figure 1</a>. Immunoblots were probed for AMPKα1 and Smc1 as control for equal loading. Data are representative of at least two independent experiments. (B and C) Thymi and secondary lymphoid organs were isolated from AMPKα1^fl/fl CD4Cre^neg and CD4Cre^pos mice and analysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone-0060217-g001" target="_blank">Figure 1</a>. Quantification of cell numbers in thymi (B) and spleens (C) are also shown. Histograms are representative for at least three mice in panels B and C. Numeric dot plots and bar graphs summarise data from at least five to six mice. Mice were analysed between 60–80 days of age. (D) iNKT cells were identified as CD24^low CD1d-αGalCer^pos thymocytes isolated from AMPKα1^fl/fl CD4Cre^neg or CD4Cre^pos thymi. Flow cytometric histograms and frequencies shown are representative of at least three independent experiments. (E) TCRβ^pos and TCRγδ^pos T cells were identified from cell preparations isolated from the epithelial layer of small intestines from AMPKα1^fl/fl CD4Cre^neg and CD4Cre^pos mice followed by flow cytometric analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060217#pone-0060217-g002" target="_blank">Figure 2</a>. Dot plot summarises frequencies of TCRβ^pos IEL cells from four different mice. Histograms are representative of four independent analyses.</p

AMPK is not required for T cell development in a TCR-transgenic model.

<p>(A) Blood biopsies were immune-phenotyped for the co-expression of the Vα2 TCR chain and CD8 and the frequency of Vα2^pos CD8^pos cells was determined in OT-1 TCR^pos AMPKα1^fl/fl CD4Cre^neg and CD4Cre^pos mice (n = 6). (B) Equal numbers of CD8^pos OT-1 TCR^pos AMPK^fl/fl CD4Cre^neg and CD4Cre^pos 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.</p

AMPKα1:a glucose sensor that controls CD8 T-cell memory

The adenosine monophosphate-activated protein kinase (AMPK) is activated by antigen receptor signals and energy stress in T cells. In many cell types, AMPK can maintain energy homeostasis and can enforce quiescence to limit energy demands. We consequently evaluated the importance of AMPK for controlling the transition of metabolically active effector CD8 T lymphocytes to the metabolically quiescent catabolic memory T cells during the contraction phase of the immune response. We show that AMPKα1 activates rapidly in response to the metabolic stress caused by glucose deprivation of CD8 cytotoxic T lymphocytes (CTLs). Moreover, AMPKα1 restrains mammalian target of rapamycin complex 1 activity under conditions of glucose stress. AMPKα1 activity is dispensable for proliferation and differentiation of CTLs. However, AMPKα1 is required for in vivo survival of CTLs following withdrawal of immune stimulation. AMPKα1(null) T cells also show a striking defect in their ability to generate memory CD8 T-cell responses during Listeria monocytogenes infection. These results show that AMPKα1 monitors energy stress in CTLs and controls CD8 T-cell memory