Event Abstract Back to Event Site-wide quantitative phosphoproteomics reveal non-redundant pathways regulated by IL-7 and IL-15 in memory CD8+ T cells Marlene J. Carrasco-Alfonso1*, Scott Ficarro2, 3, Yi Zhang2, 3, Wenyu Jiang1, Jarrod Marto2, 3 and John Luckey1* 1 Brigham and Women’s Hospital, Harvard Medical School, Department of Pathology, United States 2 Dana Farber Cancer Institute, Department of Cancer Biology and Blais Proteomics Center, United States 3 Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, United States Self-renewing memory CD8+ T-cells require IL-7 and IL-15, which preferentially promote survival versus cell growth/division. Although both signal through a common gamma-chain and JAK/STAT5, their non-redundant networks are unknown. To identify specific signaling components, we measured in highly purified primary memory-phenotype (CD44high) lymphocytes, the differential phosphorylation events downstream of IL-7 or IL-15 after equipotent stimulation ex vivo. Since conventional mass spectrometry (MS) demands samples in the range of mg, we faced the challenge of robust and large-scale quantification of post-translational modifications in primary cells. This was overcome with our automated online liquid chromatography-MS/MS platform combining nanoflow electrospray ionization with a novel 3D fractionation. By enabling examination of minute protein amounts (~50 μg/condition), we efficiently (>8000 unique phosphopeptides) completed a quantitative high-throughput site-wide (Ser-/Thr-/Tyr) phosphoproteome profiling. Notably, despite the extreme biological variability that hinders these analyses, we succeeded in detecting selected peptides (mapped to 24 proteins) that in cells from independent animal pools were consistently differentially phosphorylated (by >2-fold). This led to reveal IL-15-induced signatures: 1) AKT over-activation acting as a major determinant of cell size and proliferation (as shown by phospho-AKT/-substrate levels, and the suppression of IL-15 effects upon selective PDK1/AKT-inhibition); and 2) phosphorylation of potential effectors with unexplored functions (i.e. membrane sculpting, calcium signaling, ion homeostasis and mRNA splicing) in self-renewal. The role in IL-15-induced responses of candidate effectors that are Akt substrates is being determined. In conclusion, we provide a powerful approach for global phosphoproteomics of immune cell subsets and contribute to define the non-redundant effects of IL-7/IL-15. Keywords: CD8+ T cell memory, Interleukin-7, Interleukin-15, quantitative phosphoproteomics, AKT signaling Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Carrasco-Alfonso MJ, Ficarro S, Zhang Y, Jiang W, Marto J and Luckey J (2013). Site-wide quantitative phosphoproteomics reveal non-redundant pathways regulated by IL-7 and IL-15 in memory CD8+ T cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01014 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Marlene J Carrasco-Alfonso, Brigham and Women’s Hospital, Harvard Medical School, Department of Pathology, Boston, Massachusetts, 02115, United States, marleneca@hotmail.com Dr. John Luckey, Brigham and Women’s Hospital, Harvard Medical School, Department of Pathology, Boston, Massachusetts, 02115, United States, cluckey@partners.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marlene J Carrasco-Alfonso Scott Ficarro Yi Zhang Wenyu Jiang Jarrod Marto John Luckey Google Marlene J Carrasco-Alfonso Scott Ficarro Yi Zhang Wenyu Jiang Jarrod Marto John Luckey Google Scholar Marlene J Carrasco-Alfonso Scott Ficarro Yi Zhang Wenyu Jiang Jarrod Marto John Luckey PubMed Marlene J Carrasco-Alfonso Scott Ficarro Yi Zhang Wenyu Jiang Jarrod Marto John Luckey Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
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