Cell-to-cell communication is a fundamental process for development and maintenance of multicellular organisms. Diverse mechanisms for the exchange of molecular information between cells have been documented, such as the exchange of membrane fragments (trogocytosis), formation of tunneling nanotubes (TNTs) and release of microvesicles (MVs). In this study we assign to Fas signalling a pivotal role for intercellular communication in CD4+ T cells. Binding of membrane-bound FasL to Fas expressing target cells triggers a well-characterized pro-apoptotic signalling cascade. However, our results, pairing up flow cytometric studies with confocal microscopy data, highlight a new social dimension for Fas/FasL interactions between CD4+ T cells. Indeed, FasL enhances the formation of cell conjugates (8 fold of increase) in an early time-frame of stimulation (30 min), and this phenomenon appears to be a crucial step to prime intercellular communication. Our findings show that this communication mainly proceeds along a cytosolic material exchange (ratio of exchange >10, calculated as ratio of stimulated cells signal divided by that recorded in control cells) via TNTs and MVs release. In particular, inhibition of TNTs genesis by pharmacological agents (Latruculin A and Nocodazole) markedly reduced this exchange (inhibition percentage: >40% and >50% respectively), suggesting a key role for TNTs in CD4+ T cells communication. Although MVs are present in supernatants from PHA-activated T cells, Fas treatment also leads to a significant increase in the amount of released MVs. In fact, the co-culture performed between MVs and untreated cells highlights a higher presence of MVs in the medium (1.4 fold of increase) and a significant MVs uptake (6 fold of increase) by untreated T lymphocytes. We conclude that Fas signalling induces intercellular communication in CD4+ T cells by different mechanisms that seem to start concomitantly with the main pathway (programmed cell death) promoted by FasL.
References
[1]
Gurke S, Barroso JF, Gerdes HH (2008) The art of cellular communication: tunneling nanotubes bridge the divide. Histochem Cell Biol 129: 539–550.
[2]
Sudhof TC (2004) The synaptic vesicle cycle. Annu Rev Neurosci 27: 509–547.
[3]
Joly E, Hudrisier D (2003) What is trogocytosis and what is its purpose? Nat Immunol 4: 815.
[4]
Hudrisier D, Aucher A, Puaux AL, Bordier C, Joly E (2007) Capture of target cell membrane components via trogocytosis is triggered by a select set of surface molecules on T or B cells. J Immunol 178: 3637–3647.
[5]
Lorber MI, Loken MR, Stall AM, Fitch FW (1982) I-A antigens on cloned alloreactive murine T lymphocytes are acquired passively. J Immunol 128: 2798–2803.
[6]
Hwang I, Huang JF, Kishimoto H, Brunmark A, Peterson PA, et al. (2000) T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells. J Exp Med 191: 1137–1148.
[7]
LeMaoult J, Caumartin J, Carosella ED (2007) Exchanges of membrane patches (trogocytosis) split theoretical and actual functions of immune cells. Hum Immunol 68: 240–243.
[8]
Stinchcombe JC, Bossi G, Booth S, Griffiths GM (2001) The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15: 751–761.
Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, et al. (2008) Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 trasmission. Nat Cell Biol 10: 211–219.
[11]
Watkins SC, Salter RD (2005) Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity 23: 309–318.
[12]
Hwang I, Shen X, Sprent J (2003) Direct stimulation of na？ve T cells by membrane vesicles from antigen-presenting cells: distinct roles for CD54 and B7 molecules. Proc Natl Acad Sci USA 100: 6670–6675.
[13]
Davis DM (2007) Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol 7: 238–243.
[14]
Rustom A, Saffrich R, Markovic I, Walther P, Gerdes HH (2004) Nanotubular highways for intercellular organelle transport. Science 303: 1007–1010.
[15]
Dimitrov DS, Willey RL, Sato H, Chang LJ, Blumenthal R, et al. (1993) Quantitation of human immunodeficiency virus type 1 infection kinetics. J Virol 67: 2182–2190.
[16]
Sourisseau M, Sol-Foulon N, Porrot F, Blanchet F, Schwartz O (2007) Inefficient Human Immunodeficiency Virus Replication in Mobile Lymphocytes. J Virol 81: 1000–1012.
[17]
Onfelt B, Nedvetzki S, Benninger RKP, Purbhoo MA, Sowinski S, et al. (2006) Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J Immunol 177: 8476–8483.
[18]
Veranic P, Lokar M, Schutz GJ, Weghuber J, Wieser S, et al. (2008) Different types of cell-to-cell connections mediated by nanotubular structures. Biophys J 95: 4416–4425.
[19]
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9: 581–593.
[20]
Belting M, Wittrup A (2008) Nanotubes, exosomes, and nucleic acid binding peptides provide novel mechanisms of intercellular communication in eukaryotic cells: implications in health and disease. J Cell Biol 183: 1187–1191.
[21]
Quesenberry PJ, Aliotta JM (2010) Cellular phenotype switching and microvesicles. Adv Drug Deliver Rev 62: 1141–1148.
[22]
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, et al. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9: 654–659.
[23]
Trauth BC, Klas C, Peters AM, Matzku S, Moller P, et al. (1989) Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245: 301–305.
[24]
Zhang J, Xu X, Liu Y (2004) Activation-induced cell death in T cells and autoimmunity. Cell Mol Immunol 1: 186–192.
[25]
Kassahn D, Nachbur U, Conus S, Micheau O, Schneider P, et al. (2009) Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells. Cell Death Differ 16: 115–124.
Wang Y, Cui J, Sun X, Zhang Y (2010) Tunneling-nanotube development in astrocytes depends on p53 activation. Cell Death Differ 18: 732–742.
[28]
Arkwright PD, Luchetti F, Tour J, Roberts C, Ayub R, et al. (2010) Fas stimulation of T lymphocytes promotes rapid intercellular exchange of death signals via membrane nanotubes. Cell Res 20: 72–88.
[29]
Daubeuf S, Bordier C, Hudrisier D, Joly E (2009) Suitability of various membrane lipophilic probes for the detection of trogocytosis by flow cytometry. Cytom Part A 75: 380–389.
[30]
Puaux AL, Campanaud J, Salles A, Préville X, Timmerman B, et al. (2006) A very rapid and simple assay based on trogocytosis to detect and measure specific T and B cell reactivity by flow cytometry. Eur J Immunol 36: 779–788.
[31]
Poupot M, Fournié JJ (2003) Spontaneous membrane transfer through homotypic synapses between lymphoma cells. J Immunol 171: 2517–2523.
[32]
Canonico B, Luchetti F, Arcangeletti M, Guescini M, Degli Esposti M, et al. (2012) Flow Cytometric analyses disclose intercellular communications in FasL-stimulated T cells: results and troubleshooting. Cytom Part A 81A: 5–8.
[33]
Aucher A, Magdeleine E, Joly E, Hudrisier D (2008) Capture of plasma membrane fragments from target cells by trogocytosis requires signalling in T cells but not in B cells. Blood 111: 5621–5628.
[34]
Daubeuf S, Aucher A, Bordier C, Salles A, Serre L, et al. (2010) Preferential transfer of certain plasma proteins onto T and B cells by trogocytosis. Plos One 5: e8716.
[35]
Fulcher D, Wong S (1999) Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory. Immunol Cell Biol 77: 559–564.
[36]
Davis DM, Sowinski S (2008) Membrane nanotubes: dynamic long-distance connections between animal cells. Nat Rev Mol Cell Biol 9: 431–436.
[37]
Fukuda M (1991) Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J Biol Chem 266: 21327–21330.
[38]
Herr I, Posovszky C, Di Marzio L, Cifone MG, Boehler T, et al. (2000) Autoamplication of apoptosis following ligation of CD95-L, TRAIL and TNF-a. Oncogene 19: 4255–4262.
[39]
Waschbish A, Meuth SG, Hermann AM, Wrobel B, Schwab N, et al. (2009) Intercellular exchanges of membrane fragments (trogocytosis) between human muscle cells and immune cells: A potential mechanism for the modulation of muscular immune responses. J Neuroimmunol 209: 131–138.
[40]
Domhan S, Ma L, Tai A, Anaya Z, Beheshti A, et al. (2011) Intercellular Communication by Exchange of Cytoplasmic Material via Tunneling Nano-Tube Like Structures in Primary Human Renal Epithelial Cells. Plos One 6: e21283.
[41]
Sowinski S, Alakoskela JM, Jolly C, Davis DM (2010) Optimized methods for imaging membrane nanotubes between T cells and trafficking of HIV-1. Methods 53: 27–33.
[42]
Lee TH, D'Asti E, Magnus N, Al-Nedawi K, Meehan B, et al. (2011) Microvesicles as mediators of intercellular communication in cancer-the emerging science of cellular ‘debris’. Semin Immunophatol 33: 455–467.
[43]
Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, et al. (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68: 2667–2688.
[44]
Sarkar A, Mitra S, Mehta S, Raices R, Wewers MD (2009) Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1. Plos One 4: e7140.
[45]
Blott EJ, Bossi G, Clark R, Zvelebil M, Griffiths GM (2001) Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail. J Cell Sci 114: 2405–2416.
[46]
Süss G, Shortman K (1996) A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med 183: 1789–1796.
[47]
Daubeuf S, Puaux AL, Joly E, Hudrisier D (2006) A simple trogocytosis-based method to detect, quantify, characterize and purify antigen-specific live lymphocytes by flow cytometry, via their capture of membrane fragments from antigen-presenting cells. Nat Protoc 1: 2536–2542.
[48]
Luchetti F, Betti M, Canonico B, Arcangeletti M, Ferri P, et al. (2009) ERK MAPK activation mediates the antiapoptotic signaling of melatonin in UVB-stressed U937cells. Free Rad Biol Med 46: 339–351.
[49]
Guescini M, Guidolin D, Vallorani L, Casadei L, Gioacchini AM, et al. (2010) C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction. Exp Cell Res 316: 1977–1984.
[50]
Canonico B, Betti M, Luchetti F, Battistelli M, Falcieri E, et al. (2010) Flow cytometric profiles, biomolecular and morphological aspects of transfixed leukocytes red cells. Cytom Part B-Clin Cy 78: 267–278.
[51]
Ouasti S, Matarrese P, Paddon R, Khosravi-Far R, Sorice M, et al. (2007) Death receptor ligation triggers membrane scrambling between Golgi and mitochondria. Cell death Differ 14: 453–461.
