Phosphoproteomic analyses of IL-2 signaling reveals integrated JAK-dependent and independent phosphorylation networks that drive cytotoxic T cell fate
ABSTRACT: High-resolution mass spectrometry analysis of Interleukin 2 (IL-2) and Janus kinase (JAK) controlled protein phosphorylations in cytotoxic T lymphocytes (CTL) revealed JAKs coupled IL-2 receptors to diverse and complex serine/threonine kinase-substrate networks. These involved intricate, co-ordinated phosphorylation of transcription factors, chromatin regulators within the nuclear environment, cytosolic mRNA translational machinery, regulators of GTPases, vesicle trafficking proteins and the actin and microtubule cytoskeleton. We also identified an IL-2-JAK independent SRC family Tyr kinase controlled signaling network that regulates ~10% of the CTL phosphoproteome. One key signaling pathway in CTL is mediated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the serine/threonine kinase AKT. Strikingly, SRC family kinase dependent but JAK independent signaling controlled PIP3 levels and AKT activity in CTL. IL-2-JAK controlled signaling pathways thus coordinate with IL-2 independent networks of protein phosphorylation to program CTL fate.
Project description:Here we present high-resolution mass spectrometry analysis of changes to the phosphoproteome of cytotoxic T lymphocytes (CTL) induced by the cytokine, Interleukin 12 (IL-12). We treated CTL with a short and long IL-12 stimulation and mapped the changes to the phosphoproteome using quantitative SILAC-based phosphoproteomics.
Project description:Interleukin-2 (IL-2) and Janus kinases (JAKs) regulate transcriptional programs and protein synthesis to control the differentiation of effector CD8+ cytotoxic T cells (CTL). Using high-resolution mass spectrometry, we have generated an in-depth characterisation of how IL-2 and JAKs configure the CTL proteome to control CTL function. We found that IL-2-JAK1/3 signaling selectively regulated the abundance of a key subset of proteins influencing the accumulation of critical cytokines and effector molecules in T cells. Moreover, IL-2 controlled the concentration of proteins that support core metabolic processes essential for cellular fitness. One fundamental insight was the dominant role for IL-2 in controlling how effector T cells respond to their microenvironment. IL-2-JAK1/3 signaling pathways thus controlled the abundance of nutrient transporters, nutrient sensors and critical oxygen sensing molecules. The data provide key insights of how IL-2 controls T cell function and highlight signaling mechanisms and transcription factors that link oxygen sensing to transcriptional control of CD8+ T cell differentiation.
Project description:T Cell stretch-enhancers are vulnerable to Jak inhibitor tofacitinib Treatment of T cells with the Janus kinase (JAK) inhibitor, tofacitinib, disproportionately altered the expression of RA risk genes with stretech-enhancer (SE) structures.
Project description:Comparison of transcriptional profile of CD8 cytotoxic T lymphocytes terated with the mTORC1 inhibitor rapamycin or the mTOR inhibitor KU-0063794 and comparison with proteomic analysis. Abstract: High resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTL. We show that the CTL proteome is dominated by metabolic regulators and granzymes and that mTORC1 selectively represses and promotes expression of a protein subset (~10%) including key CTL effector molecules and signaling proteins. mTORC1 also controlled flux through a subset of metabolic pathways rather than acting as an on/off switch for global CTL metabolism. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production in CTL. Further work revealed that mTORC1 represses PIP3 production and determines the mTORC2 requirement for activation of the serine/threonine kinase AKT. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function. Overall design: Spleens from three biological replicates (control and drug treated samples were generated from the same spleens) from P14 TCR transgenic mice were harvested and activated with the antigenic peptide gp33-41 and IL-2/12 for 48h. After activation, cells were further clonally expanded in the presence of IL-2/12 for a further 96h prior to RNA extraction and hybridization to Affymetrix microarray.
Project description:In this study we compared the effects of IL-2, IL-15, and IL-21 on the gene expression, activation of cell signaling pathways, and functional properties of cells derived from the CD4+ cutaneous T-cell lymphoma (CTCL). Whereas both IL-2 and IL-15 that signal through receptors that share the common gamma chain and the beta chain modulated the expression of >1,000 genes, IL-21 that signals via the receptor also containing gamma chain up-regulated <40 genes. All three cytokines induced tyrosine phosphorylation of Jak1 and Jak3. However, only IL-2 and IL-15 strongly activated STAT5, PI3K/Akt, and MEK/ERK signaling pathways. In contrast, IL-21 selectively activated STAT3. Whereas all three cytokines protected CTCL cells from apoptosis, only IL-2 and IL-15 promoted their proliferation. The effects of the cytokine stimulation were Jak3- and Jak1-kinase dependent. These findings document the vastly different impact of IL-2 and IL-15 vs. IL-21 on malignant CD4+ T cells. They also suggest two novel therapeutic approaches to CTCL and, possibly, other CD4+ T cell lymphomas: inhibition of the Jak1/Jak3 kinase complex and, given the known strong immunostimulatory properties of IL-21 on CD8+ T, NK, and B cells, application of this cytokine to boost an immune response against malignant CD4+ T cells. Experiment Overall Design: Sez-4 cell line was starved of IL-2 for 16h, washed twice and placed into 6-well plates in 10ml RPMI (10% FBS) for 2h followed by pre-treating for 30â?? with pan-Jak inhibitor (300 nM), Jak3 inhibitor (300 nM), or drig solvent and cultured with IL-2 (200U) or medium alone for 4 h.
Project description:Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to impaired expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that repeated short-term co-cultures of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-1 (26-35)-specific CTL led to the generation of clones resistant to CTL-mediated cell death. To determine which of the UPS components and its associated pathways was responsible for CTL escape; three UKRV-Mel-15a clones were subjected to microarray gene expression analysis. Three UKRV-Mel-15a-derived melanoma clones were isolated following three repeated short-term exposures to Melan-A/MART (26-35) CTL and harvested for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation. Comparison of control and Dicer knock-out CTLs differentiated in vitro; Comparison of wild type CTLs differentiated in vitro with or without inflammatory stimuli; Comparison of effector and memory precursor CTLs isolated from mice infected with LCMV-Armstrong
Project description:Transcriptional profiling of SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) and control SAS cells (transfected with pLKO.1 vector, designated as CTL). Goal was to determine the effects of LYRIC knockdown on global SAS cells gene expression. Two-condition experiment, SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) v.s. control SAS cells (transfected with pLKO.1 vector, designated as CTL). Biological replicates: 4 control replicates, 4 transfected replicates.
Project description:Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC). In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signalling pathway, such as IL23R, JAK2 and STAT3, have been characterised, but elements unique to this network as compared to other interleukins have not been readily addressed. In this study, we have undertaken a multi-stage phosphoproteomics approach to better characterise downstream signalling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphor-enrichment approaches by maximising the capture of phosphorylation events. A total of 8069 unique phosphopeptides (containing 8344 unique sites), and 4317 unique phosphorylated proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 clearly showed induction of specific phosphorylation sites that were not readily observed after IL-2 stimulation. Interestingly, we observed subsequent translocation of STAT3 and PKM2 to the nucleus as well as increased phosphorylation especially of the nuclear portion at ~30 min after IL-23 stimulation, suggesting a wide range of cellular responses including proliferation and metabolic adaptation.
Project description:The aim of this study was to compare the signaling cascades initiated by the two closely related cytokines IL-2 and IL-15. Considering the relevance of protein tyrosine phosphorylation in signal transduction, in order to quantify changes in protein phosphorylation in response to IL-2 and IL-15, we combined triple SILAClabeling of leukemic T-cells with phosphotyrosine (pY)-specific antibody-based protein enrichment followed by mass spectrometry (MS) analysis. Following the strategy described above, we managed to decipher in detail the complex signaling networks triggered by IL-2 and IL-15. Interestingly, a large number of components of the three main signaling pathways known to be initiated in response to the interleukins (JAK/STAT; RAS/MAPK; PI3K/AKT) were identified.