Proteome Analysis of Distinct Developmental Stages of Human Natural Killer (NK) Cells
2013; Elsevier BV; Volume: 12; Issue: 5 Linguagem: Inglês
10.1074/mcp.m112.024596
ISSN1535-9484
AutoresMaxi Scheiter, Ulrike Lau, Marco van Ham, Björn Bulitta, Lothar Gröbe, Henk Garritsen, Frank Klawonn, Sebastian König, Lothar Jänsch,
Tópico(s)Immune cells in cancer
ResumoThe recent Natural Killer (NK) cell maturation model postulates that CD34+ hematopoietic stem cells (HSC) first develop into CD56bright NK cells, then into CD56dimCD57− and finally into terminally maturated CD56dimCD57+. The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56bright NK cells versus CD56dim NK cells and CD56dimCD57− NK cells versus CD56dimCD57+ NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions.Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa. The recent Natural Killer (NK) cell maturation model postulates that CD34+ hematopoietic stem cells (HSC) first develop into CD56bright NK cells, then into CD56dimCD57− and finally into terminally maturated CD56dimCD57+. The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56bright NK cells versus CD56dim NK cells and CD56dimCD57− NK cells versus CD56dimCD57+ NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa. Natural killer (NK) 1The abbreviations used are:NKnatural killerCD56NK cell markerNCAM1neural cell adhesion moleculeCD57senescence marker in T and NK cells (HNK-1 or Leu-7)CMVcytomegalovirusCPDA-1anticoagulant, containing citric acid, sodium citrate, monobasic sodium phosphate and dextroseCTLscytotoxic T lymphocytesFDRfalse discovery rateHLAself-human leukocyte antigenHSChematopoietic stem celliTRAQisobaric tags for relative and absolute quantification in mass spectrometryKIRkiller immunoglobulin-related receptors in NK cellsLC-MS/MSliquid-chromatography coupled with peptide sequencing (mass spectrometry)MADmedian absolute deviation from the medianNKISNK cell immune synapseRFregulation factor. 1The abbreviations used are:NKnatural killerCD56NK cell markerNCAM1neural cell adhesion moleculeCD57senescence marker in T and NK cells (HNK-1 or Leu-7)CMVcytomegalovirusCPDA-1anticoagulant, containing citric acid, sodium citrate, monobasic sodium phosphate and dextroseCTLscytotoxic T lymphocytesFDRfalse discovery rateHLAself-human leukocyte antigenHSChematopoietic stem celliTRAQisobaric tags for relative and absolute quantification in mass spectrometryKIRkiller immunoglobulin-related receptors in NK cellsLC-MS/MSliquid-chromatography coupled with peptide sequencing (mass spectrometry)MADmedian absolute deviation from the medianNKISNK cell immune synapseRFregulation factor. cells are large granular lymphocytes that provide a first innate immune defense. They are able to kill virus-infected and transformed cells and furthermore release cytokines and chemokines to activate adaptive immune cells (1Lanier L.L. NK cell recognition.Annu. Rev. Immunol. 2005; 23: 225-274Crossref PubMed Scopus (2256) Google Scholar, 2Vivier E. Tomasello E. Baratin M. Walzer T. Ugolini S. Functions of natural killer cells.Nat. 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The balance of signals from activating and inhibitory NK cell surface receptors tightly regulates NK cell activity. Activated NK cells release lytic granules through a process called degranulation. Therefore, NK cell cytotoxicity requires the formation of the F-actin-rich NK immune synapse (NKIS) and the transport of Perforin-containing lytic granules to the NKIS. Furthermore, this process requires granule-associated MYH9 protein (non-muscle Myosin IIa) mediating the interaction of granules with F-actin at the NKIS (3Sanborn K.B. Rak G.D. Maru S.Y. Demers K. Difeo A. Martignetti J.A. Betts M.R. Favier R. Banerjee P.P. Orange J.S. Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse.J. Immunol. 2009; 182: 6969-6984Crossref PubMed Scopus (78) Google Scholar, 4Sanborn K.B. Mace E.M. Rak G.D. Difeo A. Martignetti J.A. Pecci A. Bussel J.B. Favier R. Orange J.S. Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity.Blood. 2011; 118: 5862-5871Crossref PubMed Scopus (43) Google Scholar, 5Andzelm M.M. Chen X. Krzewski K. Orange J.S. Strominger J.L. Myosin IIA is required for cytolytic granule exocytosis in human NK cells.J. Exp. Med. 2007; 204: 2285-2291Crossref PubMed Scopus (104) Google Scholar), leading to lytic granule exocytosis. Whereas related phenotypes and functional properties are well characterized, the underlying regulatory protein network mediating differentiation, cytokine release, and cytotoxicity, is still incomplete. NK cells are defined by the expression of the surface molecule CD56 (NCAM1) and the absence of the T cell receptor (TCR) associated protein CD3 and can be further subdivided into subsets (6Fehniger T.A. Cooper M.A. Nuovo G.J. Cella M. Facchetti F. Colonna M. Caligiuri M.A. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity.Blood. 2003; 101: 3052-3057Crossref PubMed Scopus (676) Google Scholar, 7Cooper M.A. Fehniger T.A. Turner S.C. Chen K.S. Ghaheri B.A. Ghayur T. Carson W.E. Caligiuri M.A. Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset.Blood. 2001; 97: 3146-3151Crossref PubMed Scopus (1057) Google Scholar). CD56 expressing cells originate from CD34+ HSCs. Notably, the commitment to the NK lineage includes discrete steps from HSC to cells, expressing high CD56 levels (CD56bright) (8Freud A.G. Caligiuri M.A. Human natural killer cell development.Immunol. Rev. 2006; 214: 56-72Crossref PubMed Scopus (358) Google Scholar, 9Freud A.G. Becknell B. Roychowdhury S. Mao H.C. Ferketich A.K. Nuovo G.J. Hughes T.L. Marburger T.B. Sung J. Baiocchi R.A. Guimond M. Caligiuri M.A. A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells.Immunity. 2005; 22: 295-304Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), which act immune regulatory by the release of various cytokines. NK cells with low CD56-expression (CD56dim) predominantly constitute cytotoxic responses (10De Maria A. Bozzano F. Cantoni C. Moretta L. Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 728-732Crossref PubMed Scopus (280) Google Scholar, 11Poli A. Michel T. Thérésine M. Andrès E. Hentges F. Zimmer J. CD56bright natural killer (NK) cells: an important NK cell subset.Immunology. 2009; 126: 458-465Crossref PubMed Scopus (556) Google Scholar). Contact of CD56 (NCAM1) with fibroblasts (12Chan A. Hong D.L. Atzberger A. Kollnberger S. Filer A.D. Buckley C.D. McMichael A. Enver T. Bowness P. CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts.J. Immunol. 2007; 179: 89-94Crossref PubMed Scopus (262) Google Scholar) and neutrophils (13Jaeger B.N. Donadieu J. Cognet C. Bernat C. Ordoñez-Rueda D. Barlogis V. Mahlaoui N. Fenis A. Narni-Mancinelli E. Beaupain B. Bellanné-Chantelot C. Bajénoff M. Malissen B. Malissen M. Vivier E. Ugolini S. Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis.J. Exp. Med. 2012; 209: 565-580Crossref PubMed Scopus (171) Google Scholar) supports the differentiation process from CD56bright to CD56dim NK cells. The progression of early differentiation steps is proven by telomere length investigation (14Romagnani C. Juelke K. Falco M. Morandi B. D'Agostino A. Costa R. Ratto G. Forte G. Carrega P. Lui G. Conte R. Strowig T. Moretta A. Münz C. Thiel A. Moretta L. Ferlazzo G. CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation.J. Immunol. 2007; 178: 4947-4955Crossref PubMed Scopus (373) Google Scholar) and early presence in blood after HSC transplantation (HSCT) (14Romagnani C. Juelke K. Falco M. Morandi B. D'Agostino A. Costa R. Ratto G. Forte G. Carrega P. Lui G. Conte R. Strowig T. Moretta A. Münz C. Thiel A. Moretta L. Ferlazzo G. CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation.J. Immunol. 2007; 178: 4947-4955Crossref PubMed Scopus (373) Google Scholar, 15Björkström N.K. Riese P. Heuts F. Andersson S. Fauriat C. Ivarsson M.A. Björklund A.T. Flodström-Tullberg M. Michaëlsson J. Rottenberg M.E. Guzmán C.A. Ljunggren H.G. Malmberg K.J. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.Blood. 2010; 116: 3853-3864Crossref PubMed Scopus (522) Google Scholar). Indeed, CD56dim NK cells are able to change their phenotypic properties, which can be correlated with continued differentiation throughout their whole lifespan (15Björkström N.K. Riese P. Heuts F. Andersson S. Fauriat C. Ivarsson M.A. Björklund A.T. Flodström-Tullberg M. Michaëlsson J. Rottenberg M.E. Guzmán C.A. Ljunggren H.G. Malmberg K.J. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.Blood. 2010; 116: 3853-3864Crossref PubMed Scopus (522) Google Scholar, 16Hong H.S. Eberhard J.M. Keudel P. Bollmann B.A. Ballmaier M. Bhatnagar N. Zielinska-Skowronek M. Schmidt R.E. Meyer-Olson D. HIV infection is associated with a preferential decline in less-differentiated CD56dim CD16+ NK cells.J. Virol. 2010; 84: 1183-1188Crossref PubMed Scopus (60) Google Scholar, 17Béziat V. Descours B. Parizot C. Debré P. Vieillard V. NK cell terminal differentiation: correlated stepwise decrease of NKG2A and acquisition of KIRs.PloS One. 2010; 5: e11966Crossref PubMed Scopus (149) Google Scholar, 18Lopez-Vergès S. Milush J.M. Pandey S. York V.A. Arakawa-Hoyt J. Pircher H. Norris P.J. Nixon D.F. Lanier L.L. CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset.Blood. 2010; 116: 3865-3874Crossref PubMed Scopus (507) Google Scholar). CD57 was determined to be a senescence marker in T cells (19Abo T. Miller C.A. Balch C.M. Characterization of human granular lymphocyte subpopulations expressing HNK-1 (Leu-7) and Leu-11 antigens in the blood and lymphoid tissues from fetuses, neonates and adults.Eur. J. Immunol. 1984; 14: 616-623Crossref PubMed Scopus (116) Google Scholar). Recent studies determined CD57+ NK cells as a fully mature NK cell subset among the CD56dim NK cell population (CD56dimCD57+ and CD56dimCD57−). Furthermore, the NK cell differentiation process is characterized by a reversible loss of NKG2A in parallel with an irreversible acquisition of KIRs and CD57 (15Björkström N.K. Riese P. Heuts F. Andersson S. Fauriat C. Ivarsson M.A. Björklund A.T. Flodström-Tullberg M. Michaëlsson J. Rottenberg M.E. Guzmán C.A. Ljunggren H.G. Malmberg K.J. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.Blood. 2010; 116: 3853-3864Crossref PubMed Scopus (522) Google Scholar). Furthermore, CD57+ NK cells are characterized by a specialized phenotype that includes increased CD16- and Perforin-expression, reduced responsiveness to cytokines and decreased proliferation capacity. CD57 is mostly studied in the context of NK cell education that runs in parallel but uncoupled from NK cell differentiation (15Björkström N.K. Riese P. Heuts F. Andersson S. Fauriat C. Ivarsson M.A. Björklund A.T. Flodström-Tullberg M. Michaëlsson J. Rottenberg M.E. Guzmán C.A. Ljunggren H.G. Malmberg K.J. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.Blood. 2010; 116: 3853-3864Crossref PubMed Scopus (522) Google Scholar, 17Béziat V. Descours B. Parizot C. Debré P. Vieillard V. NK cell terminal differentiation: correlated stepwise decrease of NKG2A and acquisition of KIRs.PloS One. 2010; 5: e11966Crossref PubMed Scopus (149) Google Scholar, 18Lopez-Vergès S. Milush J.M. Pandey S. York V.A. Arakawa-Hoyt J. Pircher H. Norris P.J. Nixon D.F. Lanier L.L. CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset.Blood. 2010; 116: 3865-3874Crossref PubMed Scopus (507) Google Scholar). The NK cell education process encompasses the acquisition of activating and inhibitory surface receptors, like KIRs, which in turn interact with HLA class I ligands, e.g., during viral infections (19Abo T. Miller C.A. Balch C.M. Characterization of human granular lymphocyte subpopulations expressing HNK-1 (Leu-7) and Leu-11 antigens in the blood and lymphoid tissues from fetuses, neonates and adults.Eur. J. Immunol. 1984; 14: 616-623Crossref PubMed Scopus (116) Google Scholar, 20Brodin P. Lakshmikanth T. Johansson S. Kärre K. Höglund P. The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells.Blood. 2009; 113: 2434-2441Crossref PubMed Scopus (193) Google Scholar). CD57+ NK cells can recognize cytomegalovirus (CMV) and developed memory effects toward this virus (21Joncker N.T. Fernandez N.C. Treiner E. Vivier E. Raulet D.H. NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model.J. Immunol. 2009; 182: 4572-4580Crossref PubMed Scopus (201) Google Scholar, 22Sun J.C. Beilke J.N. Lanier L.L. Adaptive immune features of natural killer cells.Nature. 2009; 457: 557-561Crossref PubMed Scopus (1104) Google Scholar). Likewise, an expansion of the CD57+ NK cell population is observed during Hantavirus (24Björkström N.K. Lindgren T. Stoltz M. Fauriat C. Braun M. Evander M. Michaëlsson J. Malmberg K.J. Klingström J. Ahlm C. Ljunggren H.G. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus.J. Exp. Med. 2011; 208: 13-21Crossref PubMed Scopus (370) Google Scholar), Chikungunya virus (25Petitdemange C. Becquart P. Wauquier N. Béziat V. 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Madera S. Bezman N.A. Beilke J.N. Kaplan M.H. Lanier L.L. Proinflammatory cytokine signaling required for the generation of natural killer cell memory.J. Exp. Med. 2012; 209: 947-954Crossref PubMed Scopus (211) Google Scholar). Up to now the molecular network underlying CD57+ phenotypes are mostly characterized by FACS techniques and microarray analyses on the systemic level (15Björkström N.K. Riese P. Heuts F. Andersson S. Fauriat C. Ivarsson M.A. Björklund A.T. Flodström-Tullberg M. Michaëlsson J. Rottenberg M.E. Guzmán C.A. Ljunggren H.G. Malmberg K.J. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.Blood. 2010; 116: 3853-3864Crossref PubMed Scopus (522) Google Scholar, 17Béziat V. Descours B. Parizot C. Debré P. Vieillard V. NK cell terminal differentiation: correlated stepwise decrease of NKG2A and acquisition of KIRs.PloS One. 2010; 5: e11966Crossref PubMed Scopus (149) Google Scholar, 18Lopez-Vergès S. Milush J.M. Pandey S. York V.A. Arakawa-Hoyt J. Pircher H. Norris P.J. Nixon D.F. Lanier L.L. CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset.Blood. 2010; 116: 3865-3874Crossref PubMed Scopus (507) Google Scholar). Mass spectrometry can identify and quantify proteins in a global and unbiased manner, and thereby certainly contribute to the elucidation of developmental processes and the acquisition of specialized functions. Proteomic studies on human NK cells are accomplished mainly at the level of NK cell lines, because of the scarcity of primary NK cell subsets. Cytotoxicity, but not development, was studied in NK-92 and YTS NK cells by 2D-PAGE and peptide sequencing approaches pending on activating signals (28Rakkola R. Matikainen S. Nyman T.A. Proteome characterization of human NK-92 cells identifies novel IFN-alpha and IL-15 target genes.J Proteome Res. 2012; 4 (n.d.): 75-82Crossref Scopus (7) Google Scholar, 29Liu X.C. Liang H. Tian Z. Ruan Y.S. Zhang L. Chen Y. Proteomic analysis of human NK-92 cells after NK cell-mediated cytotoxicity against K562 cells.Biochemistry. 2007; 72: 716-727PubMed Google Scholar, 30Hanna J. Fitchett J. Rowe T. Daniels M. Heller M. Gonen-Gross T. Manaster E. Cho S.Y. LaBarre M.J. Mandelboim O. Proteomic analysis of human natural killer cells: insights on new potential NK immune functions.Mol Immunol. 2005; 42: 425-431Crossref PubMed Scopus (23) Google Scholar, 31Ghosh D. Lippert D. Krokhin O. Cortens J.P. Wilkins J.A. Defining the membrane proteome of NK cells.J Mass Spectrom. 2010; 45: 1-25PubMed Google Scholar, 32Van Damme P. Maurer-Stroh S. Plasman K. Van Durme J. Colaert N. Timmerman E. De Bock P.-J. Goethals M. Rousseau F. Schymkowitz J. Vandekerckhove J. Gevaert K. Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs.Mol. Cell. Proteomics. 2009; 8: 258-272Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). At least one proteome study investigates ex vivo expanded primary human NK cells, and focuses on the characterization of kinases, involved in NK cell activation (33König S. Nimtz M. Scheiter M. Ljunggren H.-G. Bryceson Y.T. Jänsch L. Kinome analysis of receptor-induced phosphorylation in human natural killer cells.PLoS One. 2012; 7: e29672Crossref PubMed Scopus (16) Google Scholar). Attempts to unravel the basics of NK cell development in mice were successful (34Sitnicka E. Early cellular pathways of mouse natural killer cell development.J. Innate Immun. 2011; 3: 329-336Crossref PubMed Scopus (5) Google Scholar) but not completely transferable to the human NK cell system because of a different set of surface receptors. Hence, several studies have contributed to our understanding of the role of surface receptors in different developmental stages, but studies targeting the regulation of intracellular proteins are still missing. In this study we characterized distinct developmental stages of primary human NK cells by proteomics. To get better insight into the molecular mechanisms of the NK cell differentiation process we comparatively analyzed freshly isolated primary CD56bright, CD56dim, CD56dimCD57− and CD56dimCD57+ NK cells by isobaric tags for relative and absolute quantification (iTRAQ)-based LC-MS/MS. We obtained relative quantitative data for more than 3400 proteins and observed a specific CD56+ NK cell core proteome. The obtained proteomic data strongly supports the current differentiation model of NK cells by highlighting strong distinctions between CD56bright and CD56dim NK cells and high similarity among CD57− and CD57+ NK cells. In addition to a significant set of anticipated and well-known proteins involved in NK cell development and effector functions, we detected also 11 novel protein candidates so far undescribed in NK cells. The expression patterns of S100A4 (UniProt accession name S10A4) and S100A6 (UniProt accession name S10A6), both belong to the family of S100 calcium binding proteins and contain two EF-hand domains, correlated with the developmental stages of cytotoxic NK cell subsets. Both proteins were recruited into the NKIS, following NK cell activation. For fluorescence-activated cell sorting (FACS), anti-CD56 (clone AF12–7H3, mouse IgG1, Miltenyi Biotec, Auburn, CA), anti-CD3 (clone HIT3a, mouse IgG1 κ, BD Bioscience), and anti-CD57 (clone TB03, mouse IgM, Miltenyi Biotec) mouse monoclonal antibodies (mAbs) were used. The following reagents were used: sodium chloride, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and Triton X-100 (Sigma), Mini Complete Protease Inhibitor Mixture Tablets (Roche), Benzonase (Merck), Trypsin (Promega, Madison, WI) and the iTRAQ Reagent Multiplex Kit (Applied Biosystems). Organic solvents, such as ethanol, methanol, and acetonitrile (ACN), were obtained from J. T. Baker Inc. For colocalization studies: anti-Perforin (mouse, deltaG9, BD Pharmingen); anti-CD107a (mouse, H4A3, BD Pharmingen); anti-CD107a (rabbit, 24170, Abcam, Cambridge, MA); Myosin IIa (nonmuscle, rabbit, Sigma Aldrich); S10A4 (directed against S100A4; mouse, NJ4F3, Abcam), and S10A6 (directed against S100A6; mouse, CACY-100, GeneTex) were used. This study was conducted in accordance with the rules of the Regional Ethics Committee of Lower Saxony, Germany and the declaration of Helsinki. Buffy coats from blood donations of healthy human volunteers who provided informed consent were obtained from the Institute for Clinical Transfusion Medicine, Klinikum Braunschweig, Germany. Blood donors' health is rigorously checked before being admitted for blood donation. This process included a national standardized questionnaire with health questions, an interview with a medical doctor and standardized laboratory tests for a) infections HIV1/2, HBV, HCV, Syphilis (serology and/or nucleic acid testing) and b) hematological cell counts. Buffy Coats were produced from whole blood donations on day 1 by using the Top & Bottom Extraction Bag System (Polymed Medical DevicesTM, Triple Blood Bag System, No. 7300; containing CPDA-1. Peripheral blood mononuclear cells (PBMCs) were isolated from these buffy coat products by Biocoll density gradient centrifugation (Biochrome AG) on day 2. PBMCs were cultured overnight in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS) gold (PAA Laboratories, Etobicoke, ON, Canada), 2 mm l-glutamine, 50 units/ml penicillin and 50 μg/ml streptomycin (all Invitrogen) at 37 °C in a humide 7.5% CO2 atmosphere. PBMCs were incubated with fluorochrome-conjugated anti-CD3, anti-CD56, and anti-CD57 monoclonal antibodies (mAbs) for 15 min at 4 °C on day 3. CD3−CD56dim and CD3−CD56bright NK cells as well as CD3−CD56dimCD57+ and CD3−CD56dimCD57− NK cells were isolated by FACS using a FACSAria II flow cytometer (BD Biosciences; Bionozzle size: 70 μm; system pressure: 70 PSI; flow rate 30,000 events/sec; laser: 561 nm with 50 mWatt for PE; 640 nm with 60 mWatt for APC; detection: APC 670/14, PE 585/15; 488 nm with 100 mW for FITC; detection with bandpass filters for PE 585/15, APC 670/15 and FITC 525/50). The purity and viability of the NK cell subsets were assessed by flow cytometry. In all experiments, the purity of the isolated NK cell subsets (CD3−CD56bright/CD56dim; CD3−CD56dimCD57+/CD57−) was higher than 96%. CD3−CD56dim/CD3−CD56bright (CD56dim versus CD56bright) and CD56dimCD57+/CD56dimCD57− (CD57+ versus CD57−) NK cells were each isolated from five individual human donors and mass spectrometry data were obtained and processed for each of the 10 donors individually. At a minimum, 0.9 × 106 NK cells were used for each MS experiment. NK cells were lysed in ice-cold lysis buffer (50 mm HEPES pH 7.5, 150 mm NaCl, protease inhibitor mixture supplemented with EDTA, 1% Triton-X100) on day 3. Protein concentrations were determined using a NanoDrop spectrophotometer (ND-1000, Peqlab, Biotechnology GmbH, Erlangen). Proteins were extracted from lysates by chloroform/methanol precipitation as described previously (35Wessel D. Flügge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3163) Google Scholar), and redissolved in dissolution buffer (50 mm TEAB). Equal amounts of protein from each NK cell subset was digested with sequencing grade modified trypsin from Promega, as recommended in a ratio of 1:50 at 37 °C on day 3 overnight. Subsequently, digestion was completed by adding a further 1 μg of trypsin for 2 h to each sample. Labeling of tryptic peptides with isobaric iTRAQ reagents was performed on day 4, according to the manufacturer's guidelines (Applied Biosystems, Foster City, CA). Peptides derived from CD56dim NK cells were labeled with iTRAQ reagent 117, those derived from CD56bright NK cells were labeled with iTRAQ reagent 115 Da. For quantitative MS analyses of peptides from CD3−CD56dimCD57+ (CD57+) NK cells, iTRAQ label 114 was used, and iTRAQ 116 for CD3−CD56dimCD57− (CD57−) NK cell peptides (supplemental Table S1; Supplement). Peptides of two different NK cell subsets (CD56bright and CD56dim or CD57− and CD57+ NK cells) were combined (1:1 ratio), vacuum dried, dissolved in 0.2% trifluoroacetic acid and desalted on self-packed LiChroprep RP-18 (Merck) SPE columns. The combined iTRAQ-labeled peptide samples were further subfractionated by strong cation exchange chromatography (SCX) on day 4 to support representative and comprehensive protein identification by LC-MS/MS. Peptides were dissolved in SCX buffer (0.065% formic acid, 25% ACN) and fractionated on a Mono SPC1.6/5 column connected to an Ettan micro-LC system (both GE Healthcare), and separated at a flow rate of 150 μl/min for 30 min with a linear gradient from 0% to 35% SCX buffer supplemented with 0.5 m potassium chloride. Fractions were collected by a microfraction collector, every minute (SunCollect). Peptide elution was monitored by an UV detector at 214 nm. Peptide-containing fractions were vacuum-dried, desalted by RP-C18 chromatography ZipTip pipette tips (Millipore) and analyzed separately by LC-MS/MS. LC-MS/MS analyses were performed with an UltiMate 3000 RSLCnano LC system (Thermo Scientific) connected to an LTQ Orbitrap Velos Fourier transform mass spectrometer (Thermo Scientific). Peptides were applied to a C18 precolumn (3-μm, Acclaim, 75 μm × 20 mm, Dionex) and washed with 0.1% TFA for 3 min at a flow rate of 6 μl/min. Subsequently, peptides were separated on a C18 analytical column (3-μm, Acclaim PepMap RSLC, 75 μm × 25 cm, Dionex) at 350 μl/min via a linear 120-min 3.7–31.3% B gradient with UPLC solvent A (0.1% formic acid in water) and UPLC solvent B (0.1% formic acid in 80% ACN). The LC system was operated with Chromeleon Software (version 6.8, Dionex). The effluent from the column was electro-sprayed (Pico Tip Emitter Needles, New Objectives) into the mass spectrometer. The mass spectrometer was controlled by Xcalibur sof
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