[Show abstract][Hide abstract] ABSTRACT: The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3,505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription and translation. In other cell types PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 09/2014; 9(9):e106710. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T lymphocyte proliferation and differentiation are controlled by signaling pathways initiated by the T cell antigen receptor. Here we explore how key serine-threonine kinases and their substrates mediate T cell signaling and coordinate T cell metabolism to meet the metabolic demands of participating in an immune response.
[Show abstract][Hide abstract] ABSTRACT: Krüppel-like factor 2 (KLF2) is a transcription factor that is highly expressed in quiescent T lymphocytes and downregulated in effector T cells. We now show that antigen receptor engagement downregulates KLF2 expression in a graded response determined by the affinity of T cell antigen receptor (TCR) ligand and the integrated activation of protein kinase B and the MAP kinases ERK1/2. The present study explores the importance of KLF2 downregulation and reveals that the loss of KLF2 controls a select portion of the CD8 effector T cell transcriptional program. In particular, KLF2 loss is required for CD8 T cells to express the inflammatory chemokine receptor CXCR3 and for maximum clonal expansion of T cells. KLF2 thus negatively controls the ability of CD8 T cells to respond to the CXCR3 ligand CXCL10. Strikingly, the KLF2 threshold for restraining expression of CXCR3 is very low and quite distinct to the KLF2 threshold for restraining T cell proliferation. KLF2 is thus an analogue (tunable) not a digital (on/off) cellular switch where the magnitude of KLF2 expression differentially modifies the T cell responses.
PLoS ONE 10/2013; 8(10):e77537. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T lymphocytes must regulate nutrient uptake to meet the metabolic demands of an immune response. Here we show that the intracellular supply of large neutral amino acids (LNAAs) in T cells was regulated by pathogens and the T cell antigen receptor (TCR). T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells. Slc7a5-null T cells were unable to metabolically reprogram in response to antigen and did not undergo clonal expansion or effector differentiation. The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc. Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.
[Show abstract][Hide abstract] ABSTRACT: The adenosine monophosphate (AMP)-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 (mTORC1) 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.
European Journal of Immunology 01/2013; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(3):e60217. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control
of glucose uptake and glycolysis. However, PI3K–Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible
factor 1) transcription factor complex. This mTORC1–HIF1 pathway is required to sustain glucose metabolism and glycolysis
in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of
glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and
adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen
sensing to transcriptional control of CD8+ T cell differentiation.
Journal of Experimental Medicine 12/2012; 209(13):2441-2453. · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PKD (protein kinase D) 2 is a serine/threonine kinase activated by diacylglycerol in response to engagement of antigen receptors in lymphocytes. To explore PKD2 regulation and function in TCR (T-cell antigen receptor) signal transduction we expressed TCR complexes with fixed affinity for self antigens in the T-cells of PKD2-null mice or mice deficient in PKD2 catalytic activity. We also developed a single cell assay to quantify PKD2 activation as T-cells respond to developmental stimuli or engagement of α/β TCR complexes in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T-cell development is thus critical to regulate thymic homoeostasis. PKD2-null T-cells that exit the thymus have a normal transcriptome, but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective, but critical, function for PKD2 in signalling by α/β mature TCR complexes in peripheral T-cells.
[Show abstract][Hide abstract] ABSTRACT: Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.
[Show abstract][Hide abstract] ABSTRACT: In cytotoxic T cells (CTL), Akt, also known as protein kinase B, is activated by the T cell antigen receptor (TCR) and the cytokine interleukin 2 (IL-2). Akt can control cell metabolism in many cell types but whether this role is important for CTL function has not been determined. Here we have shown that Akt does not mediate IL-2- or TCR-induced cell metabolic responses; rather, this role is assumed by other Akt-related kinases. There is, however, a nonredundant role for sustained and strong activation of Akt in CTL to coordinate the TCR- and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector versus memory and naive T cells. Akt is thus dispensable for metabolism, but the strength and duration of Akt activity dictates the CTL transcriptional program and determines CTL fate.
[Show abstract][Hide abstract] ABSTRACT: The transcriptional and metabolic programmes that control CD8(+) T cells are regulated by a diverse network of serine/threonine kinases. The view has been that the kinases AKT and mammalian target of rapamycin (mTOR) control T cell metabolism. Here, we challenge this paradigm and discuss an alternative role for these kinases in CD8(+) T cells, namely to control cell migration. Another emerging concept is that AMP-activated protein kinase (AMPK) family members control T cell metabolism and determine the effector versus memory fate of CD8(+) T cells. We speculate that one link between metabolism and immunological memory is provided by kinases that originally evolved to control T cell metabolism and have subsequently acquired the ability to control the expression of key transcription factors that regulate CD8(+) T cell effector function and migratory capacity.
[Show abstract][Hide abstract] ABSTRACT: Mammalian PKD (protein kinase D) isoforms have been implicated in the regulation of diverse biological processes in response to diacylglycerol and PKC (protein kinase C) signalling. To compare the functions of PKD1 and PKD2 in vivo, we generated mice deficient in either PKD1 or PKD2 enzymatic activity, via homozygous expression of PKD1(S744A/S748A) or PKD2(S707A/S711A) 'knockin' alleles. We also examined PKD2-deficient mice generated using 'gene-trap' technology. We demonstrate that, unlike PKD1, PKD2 catalytic activity is dispensable for normal embryogenesis. We also show that PKD2 is the major PKD isoform expressed in lymphoid tissues, but that PKD2 catalytic activity is not essential for the development of mature peripheral T- and B-lymphocytes. PKD2 catalytic activity is, however, required for efficient antigen receptor-induced cytokine production in T-lymphocytes and for optimal T-cell-dependent antibody responses in vivo. Our results reveal a key in vivo role for PKD2 in regulating the function of mature peripheral lymphocytes during adaptive immune responses. They also confirm the functional importance of PKC-mediated serine phosphorylation of the PKD catalytic domain for PKD activation and downstream signalling and reveal that different PKD family members have unique and non-redundant roles in vivo.
[Show abstract][Hide abstract] ABSTRACT: This study uses two independent genetic strategies to explore the requirement for phosphoinositide-dependent kinase-1 (PDK1) in the development of mature T cell populations from CD4/CD8 double-positive thymocytes. The data show that CD4/CD8 double-positive thymocytes that do not express PDK1 or express a catalytically inactive PDK1 mutant fail to produce mature invariant Vα14 NKT cells but can differentiate to conventional CD4, CD8, or regulatory T cell subsets in the thymus. The PDK1 requirement for Vα14 NKT cell development reflects that these cells require the PDK1 substrate protein kinase B to meet the metabolic demands for proliferative expansion in response to IL-15 or AgR stimulation. There is also constitutive PDK1 signaling in conventional α/β T cells that is not required for lineage commitment of these cells but fine-tunes the expression of coreceptors and adhesion molecules. Also, although PDK1 is dispensable for thymic development of conventional α/β T cells, peripheral cells are reduced substantially. This reflects a PDK1 requirement for lymphopenia-induced proliferation, a process necessary for initial population of the peripheral T cell niche in neonatal mice. PDK1 is thus indispensable for T cell developmental programs, but the timing of the PDK1 requirement is unique to different T cell subpopulations.
The Journal of Immunology 10/2010; 185(10):5973-82. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In normal T cell progenitors, phosphoinositide-dependent kinase l (PDK1)-mediated phosphorylation and activation of protein kinase B (PKB) is essential for the phosphorylation and inactivation of Foxo family transcription factors, and also controls T cell growth and proliferation. The current study has characterized the role of PDK1 in the pathology caused by deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN). PDK1 is shown to be essential for lymphomagenesis caused by deletion of PTEN in T cell progenitors. However, PTEN deletion bypasses the normal PDK1-controlled signaling pathways that determine thymocyte growth and proliferation. PDK1 does have important functions in PTEN-null thymocytes, notably to control the PKB-Foxo signaling axis and to direct the repertoire of adhesion and chemokine receptors expressed by PTEN-null T cells. The results thus provide two novel insights concerning pathological signaling caused by PTEN loss in lymphocytes. First, PTEN deletion bypasses the normal PDK1-controlled metabolic checkpoints that determine cell growth and proliferation. Second, PDK1 determines the cohort of chemokine and adhesion receptors expressed by PTEN-null cells, thereby controlling their migratory capacity.
Journal of Experimental Medicine 10/2009; 206(11):2441-54. · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of T lymphocytes in the thymus and the function of mature T cells in adaptive immune responses are choreographed by antigen receptors, costimulatory molecules, adhesion molecules, cytokines, and chemokines. These extrinsic stimuli are coupled to a diverse network of signal transduction pathways that control the transcriptional and metabolic programs that determine T-cell function. At the core of T-lymphocyte signal transduction is the regulated metabolism of inositol phospholipids and the production of two key lipid second messengers: polyunsaturated diacylglycerols (DAGs) and phosphatidylinositol (3-5) triphosphate [PI-(3-5)-P(3)]. The object of the present review is to discuss facts, controversies, and unresolved issues about DAG and PI-(3,4,5)-P(3) production in T lymphocytes and to discuss some of the serine/threonine kinases that control unique aspects of T-lymphocyte biology and coordinate T-cell participation in adaptive immune responses.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol-3-OH kinase (PI(3)K) and the nutrient sensor mTOR are evolutionarily conserved regulators of cell metabolism. Here we show that PI(3)K and mTOR determined the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. The key lymph node-homing receptors CD62L (L-selectin) and CCR7 were highly expressed on naive T lymphocytes but were downregulated after immune activation. CD62L downregulation occurred through ectodomain proteolysis and suppression of gene transcription. The p110delta subunit of PI(3)K controlled CD62L proteolysis through mitogen-activated protein kinases, whereas control of CD62L transcription by p110delta was mediated by mTOR through regulation of the transcription factor KLF2. PI(3)K-mTOR nutrient-sensing pathways also determined expression of the chemokine receptor CCR7 and regulated lymphocyte trafficking in vivo. Hence, lymphocytes use PI(3)K and mTOR to match metabolism and trafficking.
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.
The EMBO Journal 08/2007; 26(14):3441-50. · 10.75 Impact Factor