Identification of PIP5K1a-associated proteins in human Th17 cells by IP-MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Mandy McGeachy
PROVIDER: MSV000093068 | MassIVE | Mon Oct 09 10:38:00 BST 2023
REPOSITORIES: MassIVE
Similar Datasets
Project description:The aim of the study was to compare the two different costimuli anti-CD28 (costimulus 2a) and IL-2 (costimulus 2b) – in the presence of a suboptimal anti-CD3 signal (signal 1) – on the de novo activation of freshly purified human naive T cells. To this end, purified human naive T cells were co-incubated for seven days with a tumor vaccine which consisted of gamma-irradiated and Newcastle Disease Virus (NDV)-infected MCF-7 cells (a well-defined mammacarcinoma cell line). The gamma-irradiation dose was 200 Gray (Gy) to suppress the proliferation capacity of the vaccine cells (although the vaccine cells are not killed by such a procedure but still survive for several days); NDV-infection was performed at a dose of 100 hemagglutination units (HU) per 10^7 vaccine cells. The latter led – among others – to the upregulation of the viral surface molecules hemagglutinin neuraminidase (HN) and the fusion protein (F), both of which can serve for the attachment of fusion proteins delivering the activation signals to the naive T cells. The bispecific (bs) fusion proteins used in this study were of the single chain (scFv) format, binding with one arm to the viral HN molecule (for the attachment to the vaccine cells), and with the other arm either to the T cell receptor (TCR)-associated CD3 complex [as a surrogate of a major histocompatibility complex (MHC)-presented peptide recognized by the TCR] (bsHN-CD3), or to the costimulatory molecule CD28 (bsHN-CD28). Furthermore, a bispecific fusion protein containing the human interleukin (IL)-2 (bsHN-IL-2, a so-called immunocytokine) was used. The gene expression analysis of the differentially activated naive T cells revealed that in the presence of a suboptimal dose of anti-CD3 the two costimuli anti-CD28 (sample d7-S2_280705) and IL-2 (sample d7-S3_280705) led to a highly redundant pattern of gene upregulation in comparison to control samples in which naive T cells received only anti-CD3 (sample d7-S4_280705), anti-CD28 (sample d7-S5_280705) or IL-2 (sample d7-S6_280705) signals alone, or no signal at all (i.e. only vaccine cells; sample d7-S7_280705). All signals were compared to the reference sample which consisited of freshly isolated, non-activated naive T cells (sample d0-S1_280705). Global analysis of the gene expression showed that full naive T cell activation by anti-CD3/anti-CD28 and anti-CD3/IL-2 led to a significant upregulation of 76 and 71 genes, respectively (upregulation was considered to be significant if the relative gene expression was more than 5-fold than in the reference sample). However, the suboptimal anti-CD3 stimulus alone upregulated only 31 genes, the anti-CD28 stimulus alone only 6 genes, and the IL-2 stimulus alone only 22 genes. Naive T cells that reveived virus-modified vaccine cells alone upregulated 18 genes. Equally upregulated genes in anti-CD3/anti-CD28 and anti-CD3/IL-2 activated naive T cells comprised genes coding for both (i) signalling and (ii) effector molecules. Concerning signalling molecules, both costimuli induced the nuclear factor (NF)-kappa B and nuclear factor of activated T cells (NF-AT) pathway via the key enzymes protein kinase C (PKC)-theta and phospholipase C (PLC)-gamma 1, respectively. Of special note was that signalling molecules of the CD28-mediated stress-activated protein kinase (Sapk) pathway, namely Vav-1, Rac-1 and Jnk-2, were also upregulated by IL-2 costimulation. Upregulated genes coding for effector molecules could be classified into different functional groups: namely regulators of apoptosis, cell adhesion, receptors/ligands and cytokines/chemokines. Some examples of effector molecule-coding genes similarly upregulated in anti-CD3/anti-CD28 and anti-CD3/IL-2 stimulated naive T cells, respectively, were for instance the cell adhesion molecules LFA-1 and CD2, or members of the TNF and TNF receptor superfamilies such as CD27, CD70, CD40 ligand, Fas ligand, as well as TNF-alpha and TNF-beta (and their corresponding receptors). Among apoptosis-related genes, the anti-apoptotic gene bcl-XL as well as the pro-apoptotic gene caspase 9 were upregulated. The latter – together with the observed upregulation of cytotoxic T lymphocytes-associated protein (CTLA)-4 – might be interpreted as an indication of cellular events occurring seven days after stimulation which are aiming at termination of the response. Furthermore, the microarray analysis also revealed and upregulation of the receptor for interferon (IFN)-gamma inducible protein of 10 kDa (IP-10), namely CXCR3, of the IL-12 receptor beta-chain and of the IL-15 receptor alpha-chain; these are often seen in close association with T cell activation in general, with T cell proliferation and with generation of memory T cells. It is also of interest to mention the upregulation of beta-actin which is required for a re-organization of the cytoskeleton and the 40-fold upregulation of the metabolic enzyme glyceraldehye-3-phosphate dehydrogenase (GAPDH) which provides ATP necessary for the increased metabolism of the activated cells. A summary of the interesting gene subset is attached as a supplementary table (including the pairwise compared normalized data). In summary, these data suggest a synergism and cross-talk between the two costimulatory pathways mediated by anti-CD28/CD28 and IL-2/IL-2 receptor, respectively. Keywords: Human naive T cells, activation, costimulation, anti-CD28, IL-2, signal transduction, tumor vaccine, immunocytokine
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Project description:Cryopreserved human PBMCs from six donors were stimulated with anti-CD3/CD28 beads in the presence or absence of 100ng/ml IL-23 for 22 hours. RNA samples were assessed for consistency with a Bioanalyzer (Agilent) and quantified by a Nanodrop® ND 1000 Spectrophotometer (Nanodrop Technologies, USA). Expression array data was quantile normalised, detection above background statistical testing performed, comparison between activated un-stimulated versus activated IL-23 stimulated conditions performed as a paired test using the Illumina Custom differential expression algorithm (all implemented in Illumina BeadStudio GeneExpression Module v3.2). Two sample groups were compared, each with 6 biological replicates. Group 1 was stimulated with CD3CD28 antibody coated beads as a control group for 22hours. Group 2 was stimulated as the control group and treated with IL-23 100ng/ml for the same time period. Keywords: Cytokine Treatment
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