Abstract
Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease.
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Hickey WF, Osborn JP, Kirby WM (1985) Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat. Cell Immunol 91:528–535
Traugott U, Scheinberg LC, Raine CS (1985) On the presence of Ia-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation. J Neuroimmunol 8:1–14
Gold R, Linington C, Lassmann H (2006) Understanding pathogenesis and therapy of multiple sclerosis via animal models: 70 years of merits and culprits in experimental autoimmune encephalomyelitis research. Brain 129:1953–1971
Freund J, Stern ER, Pisani TM (1947) Isoallergic encephalomyelitis and radiculitis in Guinea pigs after one injection of brain and mycobacteria in water-in-oil emulsion. J Immunol 57:179–195
Ben-Nun A, Wekerle H, Cohen IR (1981) The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur J Immunol 11:195–199
Kies MW, Murphy JB, Alvord EC (1960) Fractionation of guinea pig basic proteins (MBP) with encephalitogenic properties. Fed Proc 22:216
Poduslo JF, McFarlin DE (1978) Immunogenicity of a membrane surface glycoprotein associated with central nervous system myelin. Brain Res 159:234–238
Williams RM, Lees MB, Cambi F, Macklin WB (1982) Chronic experimental allergic encephalomyelitis induced in rabbits with bovine white matter proteolipid apoprotein. J Neuropathol Exp Neurol 41:508–521
Paterson PY (1960) Transfer of allergic encephalomyelitis in rats by means of lymph node cells. J Exp Med 111:119–135
Gonatas NK, Howard JC (1974) Inhibition of experimental allergic encephalomyelitis in rats severely depleted of T cells. Science 186:839–841
Ben-Nun A, Wekerle H, Cohen IR (1981) Vaccination against autoimmune encephalomyelitis using attenuated cells of a T lymphocyte line reactive against myelin basic protein. Nature 292:60–61
Hickey WF, Hsu BL, Kimura H (1991) T lymphocyte entry into the central nervous system. J Neurosci Res 28:254–260
Hickey WF (1999) Leukocyte traffic in the central nervous system: the participants and their roles. Semin Immunol 11:125–137
Flügel A, Willem M, Berkowicz T, Wekerle H (1999) Gene transfer into CD4+ T lymphocytes: green fluorescent protein engineered, encephalitogenic T cells used to illuminate immune responses in the brain. Nat Med 5:843–847
Flügel A, Berkowicz T, Ritter T, Labeur M, Jenne D, Li Z, Ellwart J, Willem M, Lassmann H, Wekerle H (2001) Migratory activity and functional changes of green fluorescent effector T cells before and during experimental autoimmune encephalomyelitis. Immunity 14:547–560
Lyons AB, Parish CR (1994) Determination of lymphocyte division by flow cytometry. J Immunol Meth 171:131–137
Miller MJ, Wei SH, Cahalan MD, Parker I (2003) Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc Natl Acad Sci U S A 100:2604–2609
Marshall J, Molloy R, Moss GWJ, Howe JR, Hughes TE (1995) The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron 14:211–215
Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664
Kawakami N, Sakane N, Nishizawa F, Iwao M, Fukuda S, Tsujikawa K, Kohama Y, Ikawa M, Okabe M, Yamamoto H (1999) Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development. Immunol Lett 70:165–171
Lois C, Hong IJ, Pease S, Brown EJ, Baltimore D (2002) Germ line transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295:868–872
Miyawaki A, Sawano A, Kogure T (2003) Lighting up cells: labelling proteins with fluorophores. Nat Cell Biol 5:S1–S7
Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotech 17:969–973
Tomura M, Yoshida N, Tanaka J, Karasawa S, Miwa Y, Miyawaki A, Kanagawa O (2008) Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice. Proc Natl Acad Sci U S A 105:10871–10876
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Meth 2:905–909
Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci U S A 100:7075–7080
Mempel TR, Henrickson SE, von Andrian UH (2004) T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:154–159
Bartholomäus I, Kawakami N, Odoardi F, Schläger C, Miljkovic D, Ellwart JW, Klinkert WEF, Flügel-Koch C, Issekutz TB, Wekerle H, Flügel A (2009) Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 462:94–98
Shakhar G, Lindquist RL, Skokos D, Dudziak D, Huang JH, Nussenzweig MC, Dustin ML (2005) Stable T cell–dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat Immunol 6:707–714
Nimmerjahn A, Kirchoff F, Kerr JND, Helmchen F (2004) Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Med 1:31–37
Denk W, Svoboda K (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18:351–357
Masters BR, So PT (2004) Antecedents of two-photon excitation laser scanning microscopy. Microsc Res Tech 63:3–11
Denk W, Strickler JH, Webb WW (1990) 2-photon laser scanning fluorescence microscopy. Science 248:73–76
Niesner R, Andresen V, Neumann J, Spiecker H, Gunzer M (2007) The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging. Biophys J 93:2519–2529
Helmchen F, Fee MS, Tank DW, Denk W (2001) A miniature head-mounted two-photon microscope: high resolution brain imaging in freely moving animals. Neuron 31:903–912
Levene MJ, Dombeck DA, Kasischke KA, Molloy RP, Webb WW (2004) In vivo multiphoton microscopy of deep brain tissue. J Neurophysiol 91:1908–1912
Miller MJ, Wei SH, Parker I, Cahalan MD (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869–1873
Bousso P, Bhakta NR, Lewis RS, Robey E (2002) Dynamics of thymocyte–stromal cell interactions visualized by two-photon microscopy. Science 296:1876–1880
Bajénoff M, Egen JG, Koo LY, Laugier JP, Brau F, Glaichenhaus N, Germain RN (2006) Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25:989–1001
Miller MJ, Safrina O, Parker I, Cahalan MD (2004) Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes. J Exp Med 200:847–856
Worbs T, Mempel TR, Bölter J, von Andrian UH, Förster R (2007) CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo. J Exp Med 204:489–495
Hugues S, Fetler L, Bonifaz L, Helft J, Amblard F, Amigorena S (2004) Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. Nat Immunol 5:1235–1242
Bousso P, Robey E (2003) Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes. Nat Immunol 4:579–585
Miller MJ, Hejazi AS, Wei SH, Cahalan MD, Parker I (2004) T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc Natl Acad Sci U S A 101:998–1003
Boissonnas A, Fetler L, Zeelenberg IS, Hugues S, Amigorena S (2007) In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor. J Exp Med 204:345–356
Breart B, Lemaître F, Celli S, Bousso P (2008) Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J Clin Invest 118:1390–1397
Kim JV, Kang SS, Dustin ML, McGavern DB (2009) Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis. Nature 457:191–195
Tadokoro CE, Shakhar G, Shen SQ, Ding Y, Lino AC, Maraver A, Lafaille JJ, Dustin ML (2006) Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J Exp Med 203:505–511
Qi H, Egen JG, Huang AYC, Germain RN (2006) Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312:1672–1676
Qi H, Cannons JL, Klauschen F, Schwartzberg PL, Germain RN (2008) SAP-controlled T–B cell interactions underlie germinal centre formation. Nature 455:764–769
Bhakta NR, Oh DY, Lewis RS (2005) Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment. Nat Immunol 6:143–151
Wei SH, Safrina O, Yu Y, Garrod KR, Cahalan MD, Parker I (2007) Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node. J Immunol 179:1586–1594
Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285:221–227
Hailman E, Burack WR, Shaw AS, Dustin ML, Allen PM (2002) Immature CD4+CD8+ thymocytes form a multifocal immunological synapse with sustained tyrosine phosphorylation. Immunity 16:839–848
Thauland TJ, Koguchi Y, Wetzel SA, Dustin ML, Parker DC (2008) Th1 and Th2 cells form morphologically distinct immunological synapses. J Immunol 181:393–399
Lee K-H, Dinner AR, Tu C, Campi G, Raychaudhuri S, Tarma R, Sims TN, Burack WR, Wu H, Wang J, Kanagawa O, Markiewicz M, Allen PM, Dustin ML, Chakraborty AK, Shaw AS (2003) The immunological synapse balances T cell receptor signaling and degradation. Science 302:1218–1222
Kim JV, Jiang N, Tadokoro CE, Liu LP, Ransohoff RM, Lafaille JJ, Dustin ML (2010) Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites. J Immunol Meth 352:89–100
Kerfoot SM, Kubes P (2002) Overlapping roles of P-selectin and a4 integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis. J Immunol 169:1000–1006
Piccio L, Rossi B, Scarpini E, Laudanna C, Giagulli C, Issekutz AC, Vestweber D, Butcher EC, Constantin G (2002) Molecular mechanisms involved in lymphocyte recruitment in inflamed brain microvessels: critical roles for P-selectin glycoprotein ligand-1 and heterotrimeric G(i)-linked receptors. J Immunol 168:1940–1949
Engelhardt B, Ransohoff RM (2005) The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol 26:485–495
Vajkoczy P, Laschinger M, Engelhardt B (2001) a4-integrin-VCAM binding mediates G protein independent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J Clin Invest 108:557–565
Bauer M, Brakebusch C, Coisne C, Sixt M, Wekerle H, Engelhardt B, Fässler R (2009) b1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity. Proc Natl Acad Sci U S A 106:1920–1925
Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, Sarnacki S, Cumano A, Lauvau G, Geissmann F (2007) Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317:666–670
Geissmann F, Cameron TO, Sidobre S, Manlongat N, Kronenberg M, Briskin MJ, Dustin ML, Littman DR (2005) Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PLoS Biol 3:e113
Phillipson M, Heit B, Colarusso P, Liu LX, Ballantyne CM, Kubes P (2006) Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med 203:2569–2575
Engelhardt B (2006) Molecular mechanisms involved in T cell migration across the blood–brain barrier. J Neural Transm 113:477–485
Engelhardt B, Wolburg H (2004) Mini-review: transendothelial migration of leukocytes: through the front door or around the side of the house? Eur J Immunol 34:2955–2963
Kawakami N, Lassmann S, Li Z, Odoardi F, Ritter T, Ziemssen T, Klinkert WEF, Ellwart J, Bradl M, Krivacic K, Lassmann H, Ransohoff RM, Volk H-D, Wekerle H, Linington C, Flügel A (2004) The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis. J Exp Med 199:185–197
Barker CF, Billingham RE (1977) Immunologically privileged sites. Adv Immunol 25:1–54
Wong GHW, Bartlett PF, Clark-Lewis I, Battye F, Schrader JW (1984) Inducible expression of H-2 and Ia antigens on brain cells. Nature 310:688–691
Hickey WF, Kimura H (1988) Perivascular microglial cells of the CNS are bone-marrow derived and present antigen in vivo. Science 239:290–293
Bechmann I, Priller J, Kovac A, Bontert M, Wehner T, Klett FF, Bohsung J, Stuschke M, Dirnagl U, Nitsch R (2001) Immune surveillance of mouse brain perivascular spaces by blood-borne macrophages. Eur J Neurosci 14:1651–1658
Odoardi F, Kawakami N, Klinkert WEF, Wekerle H, Flügel A (2007) Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease. Proc Natl Acad Sci U S A 104:18625–18630
Reboldi A, Coisne C, Baumjohann D, Benvenuto F, Bottinelli D, Lira SA, Uccelli A, Lanzavecchia A, Engelhardt B, Sallusto F (2009) C–C chemokine receptor 6—regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE. Nat Immunol 10:514–523
Kawakami N, Nägerl UV, Odoardi F, Bonhoeffer T, Wekerle H, Flügel A (2005) Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J Exp Med 201:1805–1814
Lanzavecchia A, Sallusto F (2000) From synapses to immunological memory: the role of sustained T cell stimulation. Curr Opin Immunol 12:92–98
Donnadieu E, Revy P, Trautmann A (2001) Imaging T cell antigen recognition and comparing immunological and neuronal synapses. Immunol 103:417–425
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft SFB571-C6, SFB-TR43-B2, the Multiple Sclerosis competence network “Understand MS,” and the Hertie Foundation. We thank Dr. Hartmut Wekerle for his helpful comments. We acknowledge the secretarial assistance of Mrs. Cathy Ludwig.
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This article is published as part of the Special Issue on Immunoimaging of Immune System Function.
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Kawakami, N., Flügel, A. Knocking at the brain’s door: intravital two-photon imaging of autoreactive T cell interactions with CNS structures. Semin Immunopathol 32, 275–287 (2010). https://doi.org/10.1007/s00281-010-0216-x
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DOI: https://doi.org/10.1007/s00281-010-0216-x