Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy

Journal of Neuroimmunology 191 (2007) 86 – 97 www.elsevier.com/locate/jneuroim

Review article

Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy Alexander Flügel ⁎, Francesca Odoardi, Mikhail Nosov, Naoto Kawakami Max-Planck-Institute for Neurobiology, Martinsried, Germany Received 12 September 2007; accepted 12 September 2007

Abstract Two photon microscopy (TPM) recently emerged as optical tool for the visualization of immune processes hundreds of micrometers deep in living tissue and organs. Here we summarize recent work on exploiting this technology to study brain antigen specific T cells. These cells are the cause of Experimental Autoimmune Encephalomyelitis (EAE) an autoimmune disease model of Multiple Sclerosis. TPM studies elucidated the dynamics of the autoaggressive effector T cells in peripheral immune milieus during preclinical EAE, where the cells become reprogrammed to enter their target organ. These studies revealed an unexpectedly lively locomotion behavior of the cells interrupted only by short-lasting contacts with the local immune stroma. Live T cell behavior was furthermore studied within the acutely inflamed CNS. Two distinct migratory patterns of the T cells were found: the majority of cells (60–70%) moved fast and seemingly unhindered through the compact CNS parenchyma. The motility of the other cell fraction was highly confined. The cells swung around a fixed cell pole forming long-lasting contacts to putative local antigen presenting cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Two-photon microscopy; Autoaggressive effector T cells; Multiple Sclerosis; Experimental Autoimmune Encephalomyelitis

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Two photon microscopy, cell labeling techniques and imaging settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . TPM studies of T cell behavior in lymph nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TPM studies of (CNS) autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. T cells are the causative cell type of EAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Characteristics of encephalitogenic effector T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Activation of the encephalitogenic T cells is essential for EAE induction . . . . . . . . . . . . . . . . . . . . . . . 4.4. Biphasic CNS infiltration during EAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Encephalitogenic effector T cells assume a migratory phenotype in peripheral immune organs during prodromal EAE . 4.6. Encephalitogenic T cells within their target organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion and perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author. Max-Planck Institute for Neurobiology, Department of Neuroimmunology, Am Klopferspitz 18, 82152 Martinsried, Germany. Tel.: +49 89 8578 3551; fax: +49 89 89950 177. E-mail address: [email protected] (A. Flügel). 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.09.017

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1. Introduction Experimental Autoimmune Encephalomyelitis (EAE) serves as animal model for Multiple Sclerosis (MS). MS represents a highly variable disease with a wide spectrum of clinical symptoms, different disease courses, pathological changes and an unpredictable responsiveness to therapeutic interventions (Compston et al., 2006; Gold et al., 2006). There are numerous different EAE models available each of them reflecting certain aspects of MS (reviewed in (Wekerle et al., 1994; Wekerle and Flügel 2007)). While none of the models presents the entire variability of the human disease, there is a common denominator: the early, florid MS plaque and the acute EAE lesion display very similar pathological changes. The lesions are characterized by inflammatory infiltrates arranged around small postcapillary venules, which are composed mainly of T cells and monocytes/macrophages (Lassmann and Wekerle 1998). The similarities of the pathological picture between the animal model and the human disease suggest that at least the pathogenesis of the early phase of the diseases might be identical. Whereas the origin of MS is still unclear, EAE is indisputably caused by T cells which react against brain autoantigens. The knowledge of the behavior of these autoaggressive T cells in the course of the autoimmune disease process might, therefore, provide not only insights about the pathogenesis of the animal model EAE but also of the human disease. Here we will focus on recent studies exploring the migratory patterns and the functional state of encephalitogenic CD4+ T cells during different phases of EAE. These insights into the T cell behavior became possible by advances in optical microscopy techniques, in particular the development of two-photon microscopy (TPM). This technology combined with labeling of the T cells by genetic fluorescent markers enabled for the first time the visualization of encephalitogenic T cells within living milieus.

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of short waved light due to the inversed correlation of wave length and its energy. This is achieved by the “two photon effect”, which was detected more than 70 years ago by Maria Göppert-Mayer (reviewed in (Masters and So, 2004)). This effect occurs when photons come into such close contact, that they combine their energy to excite a fluorescent molecule. Different from CM, the tissue in TPM is not exposed to high energetic light along the entire laser beam, but just at the point of focus, i.e. a tiny spot of a volume of less than 1 fL (Fig. 1). This implies that any fluorescent emission signal can be used for detection. In CM, a pinhole is required which shades all light emitted from above or below the focal plane (Fig. 1). Furthermore, scattered light coming from the focal plane is evenly blocked by the pinhole, leading to a dramatic loss of signals in deeper areas. In TPM the scattered light can be used to

2. Two photon microscopy, cell labeling techniques and imaging settings TPM only recently developed from a tool manageable exclusively by physical technologists to a commercially available technique (reviewed in (Zipfel et al., 2003)). By now its application in biological research abruptly increased. TPM became so invaluable because of its ability to create pictures of high resolution and contrast at depth of hundreds of μM into tissue and organs of living animals. Like confocal microscopy (CM), TPM technology is based on lasers that systematically scan a tissue in all spatial directions and thereby generate a threedimensional picture of the specimen. The novelty of TPM resides in the use of long-waved lasers (700–980 nm) compared to conventional microscopy which employs light usually in the range of 350–640 nm. The crucial advantage of the long-waved light is that it penetrates deeper into tissues due to reduced scattering. Furthermore, its phototoxic and bleaching effects are much weaker than that of shorter-waved, high energetic light. In order to excite conventionally used fluorochromes, the energy of long waved light has to be enhanced to reach the energy level

Fig. 1. Principles of confocal and two-photon microscopy A) Single photon versus two-photon excitation are compared. In 1-photon excitation (upper left), a single photon is absorbed by a fluorochrome, raising an electron to an excited energy state, from which it relaxes to the electronic ground state and emits a lower energy photon. When the electron falls back a part of energy is dispersed due to nonradiative relaxation (NR); the rest is converted in emitted light (EM). Remarkably, all the fluorochromes along the way of the laser beam are excited (red filled area, lower left). For two-photon excitation (upper right) a fluorochrome is excited when two-photons are absorbed simultaneously and team up to excite the molecule. Once in the excited state, the electron shares the same fate as described for the single-photon excitation. Notably, the absorption is restricted to the meeting point of the photons and excitation of fluorochromes is restricted to the focus area (red dot, lower right, volume b1 fL). B) Difference between confocal and two-photon microscopy. In the confocal system (left) the emitted light generated from out of focus planes (dotted lines) is excluded by a pin hole set before the detectors. In the two photon system (right), the pin hole is not necessary: fluorescence is generated exclusively in the focal point.

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compose the picture as long as the photons follow the light path of the microscope. For deeper technological insights in TPM we refer to detailed reviews (Zipfel et al., 2003; Denk and Svoboda, 1997; Cahalan et al., 2003). Instrumental for the practicability of live imaging studies has been the development of suitable fluorescent markers and preparation techniques to gain access to the target organs. Chemical cellular dyes are frequently used to label ex vivo isolated immune cells, which are then re-implanted thereafter (reviewed in (Flügel and Bradl, 2001)). These cytoplasmic dyes usually carry lipophilic groups, e.g., acetate or acetoxymethyl side chains rendering them membrane permeable. Cytoplasmic esterases cleave these side chains off and thus trap the dyes within the cell cytoplasm, where they covalently bind to cytoplasmic proteins carrying appropriate reactive groups. The green fluorescent CFSE (carboxy fluorescein diacetate succinimidyl ester) or red fluorescent CMTMR (5-(and-6)-(4-chloromethyl(benzoyl) amino) tetramethylrhodamine) are frequently used for in vivo analysis of immune cells. These dyes exert a low toxicity and are quite stable. Labeled lymphocytes are detectable for several weeks following cell transfer into recipient animals. In addition, CFSE can be used to determine cell proliferation due to the progressive halving of the fluorescence intensity after each cell division (Lyons, 1999). However, the chemical dyes are not useful for long time recordings or the analysis of highly proliferating cells. For these applications, stable genetic labels are the markers of choice. “Green mice” or “green rats” express the green fluorescent protein (GFP) of the jellyfish Aequorea victoria in almost all tissues. Cells of these GFP transgenic animals are green under excitation light and can be easily identified after transplanting them into GFP - animals (reviewed in (Kawakami et al., 1999)). Alternatively, DNAintegrating vectors, e.g. retro-or lenti viruses, can be used as stable-labeling approaches (Mempel et al., 2004a,b; Flügel and Bradl, 2001). Imaging studies aim to follow biological processes, which often proceed over extended periods of time. Preparation techniques have to be applied in accordance with the anatomy and physiology. Due to the open access the inguinal and popliteal lymph nodes are commonly used for intravital imaging of immune processes in the nodes. Thereby the popliteal lymph node preparation seems advantageous due to less traumatic damage to the surrounding tissue and preservation of the lymph flow (Mempel et al., 2004a,b). Other immune sites which have been recently recorded using intravital TPM include the spleen (Wei et al., 2002; Odoardi et al., 2007) and the bone marrow (Mazo et al., 2005; Cavanagh et al., 2005). Bone marrow cells can be directly imaged within the bone marrow cavities through the thinned skull preparation. The spleen has to be exposed before by laparotomy. One obvious difficulty lies in the limited access to organs, which are located deep in the body or which are shielded by bone structures. Neurobiological studies have found a solution to prolong the observation period of brain tissue by inserting a window into the skull or the spinal cord (Helmchen and Denk 2002a; Misgeld and Kerschensteiner 2006). As a complementary approach, acute slices of brain or spinal cord can be used to study (immune) cell behavior within the living

nervous tissue (Pettit et al., 1995; Margrie et al., 2003; Kawakami et al., 2005; Nitsch et al., 2004b). Usually, long-term imaging analyses are hindered by time limits for narcosis and the onset of inflammatory processes. To date, as an alternative approach, sequential recordings of organ explants are performed. Comparative studies of intact animals and explanted lymph nodes (LNs) proved the feasibility of this approach: the motility of T cells under both conditions was virtually identical (Cahalan et al., 2002). However, it was found that the culture condition of the explanted organs is crucial. Thus, similar to brain slices, LNs as highly vascularized and metabolically active tissues need highly oxygenized buffer conditions (95% O2, 5% CO2). Initial LNs studies under cover glass with limited nutrition or lower oxygen supply revealed immediate rounding up of cells and immobilization (Wei et al., 2002; Huang et al., 2007). 3. TPM studies of T cell behavior in lymph nodes The pioneering TPM studies in the field of immunology appeared in the year 2002 by Cahalan's group exploring lymphocyte motility within LNs explants (Miller et al., 2002) and by Robey and colleagues analyzing thymocytes in a three-dimensional thymic organ culture (Bousso et al., 2002). TPM very rapidly became an essential tool to examine immune processes directly within their natural environments (reviewed in (Cahalan et al., 2003; Flügel and Kawakami, 2005; von Andrian and Mempel, 2003; Germain et al., 2006). The first TPM studies focused on the intrinsic motility of immune cells in lymph nodes. Immune cells in vivo moved similarly as observed before in vitro. Thus, in a 3D collagen matrix T cells migrated with a mean velocity of 7 μm/min reaching maximal value of 25 μm/min (Friedl et al., 1998). T cells in LNs moved with ∼10 μm/min reaching similar peak velocities of N 25 μm/min (Bousso and Robey 2003; Miller et al., 2002; Miller et al., 2003). Dendritic cells moved considerably slower in vitro than in vivo (∼2.5 μm/ min versus 6 μm/min, respectively (Friedl et al., 1998; Bousso and Robey 2003). Unexpectedly the motility of the cells within the nodes was apparently random. This was independent on the cell type and was equally observed in LN-explants and intact animals (Bousso and Robey 2003; Miller et al., 2002; Miller et al., 2003; Miller et al., 2004b; Mempel et al., 2004a). The impression of unhindered locomotion was strengthened by the observation that in 2-photon images the fluorescent cells seem to move within a black fluid. Recent studies, however, which examined cell motility together with the anatomic structures revealed that extracellular matrix scaffolds act as guidelines for the cells in lymph nodes (Bajénoff et al., 2006). Furthermore, in accordance with previous concepts (Moser et al., 2004) chemokine gradients within the nodes were found to influence the orientation (chemotaxis) (Okada et al., 2005) and velocity (chemokinesis) (Worbs et al., 2007) of the immune cells. One of the central immunological questions addressed by the TPM-studies was how antigen would be presented to T cells under real life conditions. The frequencies of T cells with specificity for a certain antigen in healthy immune repertoires are extremely low (∼ 1 in 105–106 T cells, reviewed in (Rufer

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2005)). Therefore it is hard to imagine how antigen specific T cells would find their cognate antigen within reasonable time. Two independent studies demonstrated that the chance for a T cell to meet a matching antigen presenting cell (APC) is greatly increased by rapid and short lasting scanning of the APCs (Miller et al., 2004a,b; Bousso and Robey 2003). In these studies the numbers of contacts of one individual dendritic cell (DC) with different T cells were calculated resulting in the enormous number of 500 contacts/h for CD8+ T cells (Bousso and Robey 2003) and 5000 contacts/h for CD4+ T cells (Miller et al., 2004a, b; Bousso and Robey 2003). Furthermore, encounters of T cells and DCs seem to be promoted by structural factors of the LNs. Thus, naïve T cells entering the node are guided through a network of DCs (Lindquist et al., 2004) which substantially increase the likelihood of hunting up specific antigens by the T cells (reviewed in (Cahalan and Parker 2006)). The process of antigen encounter of specific T cells in LNs is still controversial. It is known that DCs play a crucial role for antigen presentation and activation of naive T cells (Ingulli et al., 2002; Norbury et al., 2002; Schaefer et al., 2002). The duration of the encounter and the nature of the contact site between T cells and DCs is, however, still not clear. The results of in vitro studies indicated long-lasting and stable contacts formed by immunological synapses, highly structured ring-shaped arrangements of membrane molecules of the TCR/MHC activation complex and adhesion molecules, termed as supramolecular activation complexes (SMACs). (Monks et al., 1998; Dustin and Cooper 2000; Bromley et al., 2001). Other researchers postulated a “serial encounter model” characterized by short and transient interactions with varying contact sites (Valitutti and Lanzavecchia 1997; Gunzer et al., 2000; Underhill et al., 1999). Von Andrian's group introduced a three step model of antigen recognition in vivo, which combines aspects of both, stable and transient antigen encounters (Mempel et al., 2004a). This model distinguishes three temporally separated stages: in the initial 8 h following contact with antigen-loaded DCs, T cells form short serial encounters with the DCs. This phase is followed by formation of long lasting (∼20 h) stable conjugates, a period, during which T cells start to produce cytokines. After 48 h, the T cells regain their motility and proliferate. This multi-step model of antigen recognition was basically confirmed by others (Miller et al., 2004b; Hugues et al., 2004). Still, none of the groups could definitely answer the issue of stable versus serial encounters, because it remained difficult to follow single cells over time. A hint that the nature of the interaction between the T cell/APC might be relevant for the functional outcome of the T cell response came from Amigorena's group (Hugues et al., 2004), who observed that CD8+ T cells which were stimulated adequately with LPS-activated DCs or with DCs coinjected with costimulating anti-CD40 antibodies became efficient cytotoxic effector T cells. These cells went through a stadium where they formed stable contacts with the DCs. In contrast, the same T cells primed under suboptimal conditions (not activated DCs) became tolerized, and these cells exclusively formed short-lasting contacts (Hugues et al., 2004). Similar observations were made very recently with CD4+ T cells. This intravital imaging study explored the influence of the stimulatory potential of the peptide/

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MHC complex on T cell motility in lymph nodes. Motility and activation of the murine moth cytochrome C-specific T cells was tested with different peptide ligands. T cell deceleration and activation was exclusively observed with a highly stimulatory peptide, whereas low stimulatory peptides did not influence T cell locomotion and induced anergy (Skokos et al., 2007). Other questions addressed by TPM studies are the location and sequence of various cell–cell interactions during immune responses, including B cell activation (Schwickert et al., 2007; Okada et al., 2005; Allen et al., 2007), regulatory processes (Tadokoro et al., 2006; Tang et al., 2006; Mempel et al., 2006), anergy and tolerance induction (Hugues et al., 2006; Shakhar et al., 2005), and cytotoxicity against tumors (Boissonas et al., 2007; Mempel et al., 2006) or allogeneic cells. 4. TPM studies of (CNS) autoimmunity To date there are very few reports on the use of TPM for questions in autoimmunity. Bluestone's group explored the behavior of islet-specific CD4+ T cells and regulatory T cells (Treg cells) in lymph node explants of nonobese diabetic mice (Tang et al., 2006). Islet specific T cells isolated from T cell receptor transgenic mice (BDC2.5+ T cells (Katz et al., 1993)) and labeled with CFSE were transferred to either NOD or NOD. CD28−/− Treg cell deficient mice. In the inguinal lymph nodes the cells migrated to the paracortical T cell zones. The cells were found to move differently in pancreas-draining lymph nodes and not draining inguinal lymph nodes. Whereas their locomotion pattern in the inguinal lymph nodes was similar to foreign antigen-specific T cells reported before (Miller et al., 2002, 2004b; Mempel et al., 2004a; Stoll et al., 2002; Bousso and Robey 2003), their motility was significantly changed in the pancreas-draining lymph nodes: a substantial fraction of the cells formed swarms starting 18 h after T cell transfer. These cells showed restricted trajectories, reduced displacements and their average velocity was moderately reduced (form ∼ 8 μm/min to ∼6 μm/min). Transfer of the islet specific T cells into NOD. CD28−/− Treg cell deficient mice revealed an even stronger effect on the motility of the autoantigen-reactive T cells within the pancreatic nodes. A part of the cells became arrested in clusters. Their velocity was reduced to ∼ 1 μm/min (maximal decrease 18 h after T cell transfer). This behavior correlated with T cell activation and proliferation within the pancreatic nodes. Islet antigen specific BDC2.5+ Treg cells were found to prevent these changes in T cell motility and activation of the autoreactive T cells. Thereby the Treg cells seemed not to interact directly with the autoreactive effector T cells but with local APCs within pancreatic nodes (Tang et al., 2006). How did TPM contribute to the understanding of CNS autoimmunity? Before we will discuss this aspect we would like to give a brief overview of our knowledge about encephalitogenic T cells during EAE. 4.1. T cells are the causative cell type of EAE EAE evolved at the change of the 19th century as sequelae of rabies vaccination (Zamvil and Steinman, 1990). Patients

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having received Pasteur's treatment, a 1885 developed vaccine containing fixed rabies grown in rabbit brain, in 0.1% developed severe, in some cases lethal paralytic disease. A decade later, Rivers and colleagues induced demyelinating encephalomyelitis in rhesus monkeys after repeated (N 50 times) exposition with rabbit CNS (Rivers et al., 1933). From these pioneering experiments the most commonly used way to induce EAE in animals has been evolved, which is actively induced EAE, i.e. EAE provoked by active immunization against CNS structures (aEAE). The observation that immunization of brain tissue together with adjuvant, i.e. homogenized mycobacteria emulsified in mineral oil (Complete Freund's Adjuvant, CFA) strongly enhanced the immunogenicity of the inoculum and thus, reduced the number of vaccinations needed to trigger CNS inflammation, greatly facilitated EAE research (Kabat et al., 1947; Freund et al., 1947). Since then, EAE has been induced in many different species (reviewed in (Owens, 2006; ‘t Hart et al., 2005)). An important step in EAE research was the finding that brain antigen specific T lymphocytes are the cellular cause of EAE. This was ultimately proven by transferring CD4+ MBP-specific T lymphocytes into healthy syngeneic recipient animals, which thereafter developed classical EAE (tEAE) (Ben-Nun et al., 1981; Mokhtarian et al., 1984). 4.2. Characteristics of encephalitogenic effector T cells Based on the observation that CD4+ T cell populations which transfer EAE produce the pro-inflammatory cytokines IFNγ and IL-2 (Ben-Nun et al., 1981; Pettinelli and McFarlin, 1981; Kuchroo et al., 1992; Zamvil et al., 1985) encephalitogenic T cells were classically categorized as TH1 lymphocytes. However, this concept was repeatedly questioned. Murine studies using IFNγ neutralizing antibodies (Billiau et al., 1988) or which induced EAE in IFNγ-deficient mice (Krakowski and Owens 1996; Willenborg et al., 1996) revealed a paradoxical, enhancing effect on EAE in the absence of IFNγ. The discovery of CD4+ T cells (TH17 cells), which produce IL-17 as marker cytokine, rather than IFNγ might explain early findings (Langrish et al., 2005; Park et al., 2005). In rodents, these cells require a combination of TGFβ, and IL-6 for lineage decision, instead of IL-12 (Veldhoen et al., 2006; Bettelli et al., 2006b). Pathogenic T cells were commonly considered to be brainautoreactive CD4+ CD8−, αβ T cell receptor+ T lymphocytes which recognize their target peptide epitopes in context of MHC class II. In contrast, CD8+ T cells were considered to function as regulatory cells in EAE (Najafian et al., 2003; Sun et al., 1988; Abdul-Majid et al., 2003; Hu et al., 2004; Koh et al., 1992). However, several studies independently identified myelin autoreactive CD8+ T cells in C3H and C57BL/6 mice as mediators of autoimmune brain inflammation. (Huseby et al., 2001; Sun et al., 2001; Ford and Evavold, 2005). Myelin antigens are the prototype T cell antigens of EAE. T cells reactive against myelin basic protein (MBP), the most redundant myelin component, were firstly identified as highly pathogenic cells in classic tEAE models (Wekerle et al., 1994). However it soon became clear that there may be encephalito-

genic T cells directed against many other, not necessarily myelin CNS antigens, such as myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), or myelin associated glycoprotein (MAG), glial fibrillary acidic protein (GFAP) or the Ca2+-binding S100β protein (Wekerle et al., 1994). 4.3. Activation of the encephalitogenic T cells is essential for EAE induction Adequate activation of autoreactive effector T cells is crucially required to initiate EAE. Actively induced EAE models apply emulsions of brain antigens in mineral oil together with bacterial components (complete Freund's adjuvant). Bacterial components, pathogen-associated molecular patterns (PAMPs) are sensed by the APCs via toll-like receptors and other spezialized receptors. These signals induce a pro-inflammatory milieu for the initiation of effective adaptive immune responses (Medzhitov and Janeway, 2002). In transferred EAE, encephalitogenic T cells must also be strongly activated to induce clinical disease (Wekerle et al., 1986). Resting lymphocytes are unable to infiltrate the brain. One factor might be a different circulation behavior of resting compared to activated cells (Mackay et al., 1992; Bradley et al., 1999). However, the crucial difference which enables activated T cells but not resting T cells to enter the brain and induce local inflammation remains unknown. As discussed below, cytokines or membrane surface molecules which are transiently expressed primarily in the activated T lymphocyte might be essential to pre-activate the CNS tissue (Merrill and Benveniste 2000; Owens et al., 1994). 4.4. Biphasic CNS infiltration during EAE One of the open questions of EAE is how the freshly activated encephalitogenic T cells enter the CNS and induce clinical disease. This has been most extensively analyzed in transfer models of EAE, and herein, in the tEAE of the Lewis rat induced by MBP-specific T cells. This firstly established “classic” transfer EAE model is particularly suited to study the behavior of encephalitogenic T cells. Its unrivalled reproducibility and predictability allows to reliably track the pathogenic cells during the different phases of EAE. The disease is characterized by a monophasic caudo-cranially ascendent paralysis. Histologically, the EAE lesions present the characteristic mononuclear cell infiltrates dominated by monocytes and CD4+ T lymphocytes, while B cells play a minor role (Traugott et al., 1982; Trotter and Steinman, 1984). Notably in tEAE clinical disease does not start immediately after transfer of the activated T cell blasts but there is an obligatory disease free phase of at least 3–4 days. This delay seems not to be due to brain-specific migratory signals used by the encephalitogenic T cells. Thus it could never been shown that migration of these cells involves different adhesion molecules than those used in the periphery, suggesting that similar steps of lymphocyte extravasation via tethering, adhesion and transmigration take place in the brain as in the periphery. Accordingly, EAE studies involving transgenic animals or using

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blocking antibodies show participation of all commonly used members of adhesion molecule classes for lymphocyte trafficking, the selectins, integrins, chemokines, and adhesion molecules from the immunoglobulin superfamily (reviewed in (Engelhardt, 2006; Ransohoff, 1999)). The reason for the delayed T cell homing into the CNS is commonly attributed to the specific immune situation of the brain (Wekerle et al., 1986; Hickey, 1991). The CNS in the healthy state represents an immune hostile environment, as reflected by its specialized anatomical structure and functional properties. The brain is secluded from the blood circulation by the tight blood brain barrier which closely restricts the access of soluble molecules and cells. Immune cells are rare and lymphatic vessels are missing. The expression of factors needed for an efficient immune response, e.g. MHC molecules, costimulatory factors, cytokines and chemokines are extremely low. Recognition of brain antigen is, however, required for encephalitogenic CD4+ T cells in order to induce brain inflammation (Wekerle et al., 1986). Myelin antigens are produced by oligodendrocytes which do not express MHC molecules. Therefore, the antigen has to be “cross-presentated” by local APCs. The T cells arriving in the CNS in the course of tEAE are antigen-experienced effector T cell, i.e. they are less dependent on co-stimulatory signals than naïve T cells (Croft et al., 1994; Garcia et al., 1999; McKnight et al., 1994). However, can the autoimmune process get started if MHC molecules are virtually absent within the brain? According to the traditional view the autoreactive T cells solve this problem by invading into the brain twice (Wekerle et al., 1986; Hickey et al., 1991). The first T cells enter the CNS immediately after transfer (hours after transfer, p.t.). This is then followed by a second, massive inflammatory wave coinciding with onset of clinical symptoms (3–4 days p.t.). The first invaders, the “pioneers”, enter the CNS as freshly activated T cell blasts and are able to produce high amounts of pro-inflammatory cytokines independent on antigen recognition. Therefore, these cells are supposedly well equipped to create an immune permissive milieu and thus set the stage for the second inflammatory wave, which contains antigen-specific cells as well as non-specific recruited immune cells. Thereby monocytes/macrophages seem to play a major role in causing clinical disease, most likely due to their capacity to release large amounts of potentially toxic cytokines acting on neurons and glial cells (Huitinga et al., 1990). 4.5. Encephalitogenic effector T cells assume a migratory phenotype in peripheral immune organs during prodromal EAE Data about the first immune phase of pioneer T cells are sparse. This is most likely due to their low numbers. It could be shown however, that homing of these cells into the CNS depends on P-selectins (Carrithers et al., 2000), LFA-1 (Laschinger et al., 2002), and VLA-4 (Vajkoczy et al., 2001). Transmigration into the CNS parenchyma by these cells seems to follow an atypical pattern, since firm adhesion to the endothelial classic was not preceded by rolling along the endothelia (Vajkoczy et al., 2001).

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Since just few of the transferred encephalitogenic effector T cells invade the CNS as pioneer T cells, the question arises, where do the rest of the cells go? We addressed this question by inducing tEAE in the Lewis rat with retrovirally engineered, green fluorescent protein-expressing T cells, which were reactive against MBP (TMBP-GFP cells). Due to stable integration of the marker gene the cells remained detectable throughout the entire EAE course (Flügel et al., 1999). Using cytofluorometric cell analyses we found that the majority of the T cells during prodromal EAE indeed remained within peripheral organs (Flügel et al., 2001). The cells migrated on a predefined route through the secondary immune organs. Within the first 48 h they accumulated in the parathymic lymph nodes. Thereafter (72–84 h) they could be found in spleen and blood, before they invaded in masses their target organ (84–96 h). Effector T cells which did not react against brain antigens (e.g. OVA-specific T cells) showed a similar initial migratory behavior. These cells failed, however, to invade the CNS in higher numbers. Instead they distributed throughout the secondary lymph organs (Flügel et al., 2001). On their way to the CNS, encephalitogenic T cells assumed a “migratory phenotype”: they down-regulate activation markers (OX-40, IL-2 receptor) and up-regulate chemokine receptors (Flügel et al., 2001). Recent gene expression analyses of T cells isolated from the spleen 3 d after T cell transfer (p.t.) revealed that these changes were not restricted to few genes, but comprised more than 500 genes including adhesion molecule-, cytokine-, chemokine-, protease-, cell cycle-, apoptosis-, and transcription factor-genes (not published). Thus, the phenotype of autoreactive T cells within the spleen radically differed from the one of the cultured counterparts. How do these migratory effector T cells move within the peripheral milieu? We addressed this question by imaging TMBP-GFP cells in the spleen 3 d p.t. (Odoardi et al., 2007). The majority of TMBP-GFP cells (N80%) was in permanent motion, with only a few scattered T cells attached to anchoring points (Supplementary Video 1). The motile TMBP-GFP cells moved around apparently at random, as indicated by their trajectories (Fig. 2A). Their mean velocity was 7–8 μm/min with peaks of N25 μm/min. Furthermore, the cells did not move constantly, but followed a “stop-and-go” mode, i.e. phases of rapid progression alternated with phases of respite (Odoardi et al., 2007). Thus, surprisingly the migration characteristics of the encephalitogenic effector T cells was very similar to the one of murine naïve T cells in lymph nodes (Miller et al., 2002). Intravenous (i.v.) injection of MBP dramatically changed the locomotion pattern and the functional state of the effector T cells. Within 10–15 min the TMBP-GFP cells slowed down and formed durable contacts with splenic phagocytes (Fig. 2B). The cell arrest went along with strong up-regulation of membrane activation markers and production of pro-inflammatory cytokines (Odoardi et al., 2007). This immediate change of the T cell motility and activation state strongly contrasted with antigen encounters by naïve CD4+ T cells, where stable cluster formation was preceded by a period of multiple, transient T cellAPC contacts (Catron et al., 2004; Mempel et al., 2004a; Miller et al., 2004b). Furthermore, a T cell response of ∼10 min after

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introduction of antigen is unexpectedly rapid taking into consideration that a complex chain of reactions must have occurred within this short period. These include the uptake of antigenic proteins by local APCs, the processing and presentation in the proper MHC class II context, followed by antigen recognition via TCRs, and the proper response by the T cells. Soluble antigen infusion led to depletion of the effectors in the periphery. After a period of 2–3 days, during which the cells were non-responsive to their specific antigen, they underwent antigen-related cell death (Odoardi et al., 2007). Trapping of the cells had dramatic consequences on the course of EAE. The treatment totally prevented the formation of histological infiltrates and clinical EAE (Fig. 2C). These findings imply an essential role of encephalitogenic T cells not only as pioneer T cells opening the blood brain barrier, but also as effector cells during the second inflammatory wave. To confirm this and to exclude that the strong activation of the effector T cells induced by the intravenous antigen infusion would not-specifically impair the autoimmune CNS inflammation we used an alternative approach to selectively target encephalitogenic T cells in vivo.

Fig. 2. Effect of i.v MBP infusion in different phase of EAE. A) Migratory path of TMBP-GFP cells (green) in the spleen. Two-photon microscopy. 10 min videos, 30 s intervals. Yellow lines track motile TMBP-GFP (cells that moved N10 μm/10 min). Cyan dots indicate stationary TMBP-GFP cells (cells that migrated b10 μm/10 min). Scale bars: 10 μm. B) Effect of soluble antigen infusion on effector TMBP cell motility. Average velocity of TMBP cell (B) in spleen (blue diamonds) or CNS (red triangles) after MBP (filled lines) or OVA antigen (dotted lines). Two-photon microscopy. 120 min videos. Antigen was infused 60 h and 96 h after TMBP cell transfer for spleen and CNS motility analysis respectively. Arrows indicate time points from which the values significantly decreased (p b 0.05). C) Clinical effect of i.v MBP infusion after transfer of 5 × 106 TMBP-GFP cells. Antigen was infused i.v. 48 h (blue bars) or 96 h (red bars) after adoptive transfer. Black bars: not treated control animals. Arrows indicate the time point of MBP infusion. Clinical scores (y axis): 0 = no disease; 1 = flaccid tail; 2 = gait disturbance; 3 = complete hind limb paralysis; 4 = tetraparesis; and 5 = death. Note that 33% of animals treated 96 h p.t. died (asterisk).

Fig. 3. Gancyclovir treatment ameliorates clinical EAE. A) Clinical course in PBS (white) or gancyclovir (black) treated animals. The disease was induced by transfer of 5 × 106 TMBP-GFP-HSV-TK+ cells. Grey bar: period of treatment. B) Reduction of effector T cells infiltration after gancyclovir treatment. The number (± SD) of TMBP-GFP-HSV-TK+ cells in EAE lesions was quantified by cytofluorometry 4 days after transfer. White bars: control animals, black bars: gancyclovir treated animals.

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We retrovirally introduced the suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK) into the TMBP cells (M. Nosov, unpublished). Expression of HSV-TK rendered the cells sensitive to gancyclovir (GCV), a non-toxic prodrug which is converted within the cells through HSV-TK-mediated phosphorylation into cytotoxic derivative GCV triphosphate (Bonini et al., 1997). Using the marker gene of GFP transcriptionally coupled to HSV-TK gene we were able track the cells and to quantify the effect of GCVon the tagged cells in vivo. We found that GCV induced rapid death of HSV-TK+ T cells in vitro and in vivo. Elimination of autoreactive T cells early in the course of prodromal phase of EAE substantially suppressed CNS inflammation and disease induction (Fig. 3). These data indicate

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that clinical disease and the accompanying autoimmune inflammation is mediated by the migratory effector T cells and the numerical contribution of the pioneer cells to the florid infiltrations arising with onset of disease is negligible. 4.6. Encephalitogenic T cells within their target organ EAE studies tracking stably labeled encephalitogenic T cells revealed that the majority of the T cells (N 90%) which enter the CNS after the prodromal interval of 3–4 days, are autoantigen specific (Flügel et al., 2001; Ransohoff et al., 2003; Bauer et al., 1998). Millions of the post-activated effector T cells invaded their target organ. These cells were not restricted to the vessels

Fig. 4. Effector T cells in the CNS. A) Distribution of TMBP-GFP cells (green) in EAE lesions 4 days after transfer. Confocal microscopy. White matter. CNS parenchyma is labeled by Topro. Scale bars: 10 μm. B) Motility pattern of effector T cells in the target organ. Migratory paths of TMBP-GFP cells (green) in the meninges 4 days after transfer. Two-photon microscopy. 10 min intervals. The dotted lines indicate trajectories of motile (yellow) or stationary (cyan) cells. Vessels are visualized by dextran-texas red injection. Scale bar: 10 μm. C) TMBP-GFP cells form immune synapses like structures with potential antigen presenting cells in the CNS. 3Dreconstruction of TMBP-GFP cells (green) establishing contact with MHCclassII+ cells (red). Note the polarization of TCR (blue) at the contact point. Confocal microscopy on CNS tissue fixed 96 h after T cell transfer. D) TMBP cells are reactivated in the CNS after i.v. antigen infusion. Intracellular staining for IFNγ and IL17 in effector TMBP cells isolated form CNS 6 h after i.v. OVA (upper plots) or MBP infusion (lower plots), 96 h after transfer of 5 × 106 TMBP-GFP blasts in recipient Lewis rats. Cytofluorometry. Left dot plots: IgG controls.

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and the perivascular space, but they were found deep within the CNS parenchyma (Fig. 4A). As discussed above the healthy brain is almost devoid of immune cells. The compact tissue is filled with cells and their processes specialized to guarantee undisturbed nerve conduction (“glial glue”) and lacks spacious interstitial rooms. Therefore, the massive accumulation of encephalitogenic cells in early EAE lesions and their rapid penetration deeply into the CNS parenchyma raised the question how the cells would move within this compact tissue. We addressed this by examining the motility behavior of encephalitogenic TMBP-GFP cells in acutely inflamed spinal cord tissue 4 days after induction of tEAE (Kawakami et al., 2005). We analyzed the tissue on two levels. We used acute spinal cord slices to visualize the effector T cell behavior deep within the spinal cord white and grey matter (Kawakami et al., 2005). Additionally, laminectomy at the level of the lumbar spinal cord enabled us to record TMBP-GFP cells within the meningeal areas and the adjacent white matter of intact animals (Odoardi et al., in press) (Fig. 4B, Supplementary Video 2). The autoaggressive effector T cells moved seemingly unhindered and fast through the CNS with two distinct migratory patterns: about 60% of the T cells moved rapidly through the compact white and grey matter (up to 25 μm/min), while the rest of the cells were attached and moved around a fixed anchor point (Fig. 4B, Supplementary Video 2). Similarly to the movement behavior in the spleen, the motile T cells migrated rapidly along seemingly random tracks with pulsating speed. The tethered T cells spun around synapse-like concentrations of their TCR, which colocalize LFA-1 (Kawakami et al., 2005). These “synapses” touched local cells expressing MHC class II (Fig. 4C). Pretreatment of the affected tissue with neutralizing anti-MHC class II monoclonal antibodies significantly reduced the number of arrested T cells (Kawakami et al., 2005). Conversely, intravenous infusion of soluble MBP at this acute EAE phase caused rapid deceleration of the T cells (Fig. 2B). The antigen was found to diffuse through the breached blood brain barrier and to be presented locally to the autoaggressive effector T cells (Odoardi et al., in press). As a consequence, the majority of effector T cells throughout the CNS parenchyma and the meninges became arrested within 1–2 h and the expression of pro-inflammatory cytokine was strongly increased (Fig. 4D). The clinical outcome after i.v. antigen during the acute disease phase of EAE dramatically differed from the one of the preclinical treatment (48 h p.t.): the disease was significantly worsened (Fig. 2C) (Odoardi et al., in press). 5. Conclusion and perspectives TPM technology has substantially increased our knowledge of the dynamics and the sequence of autoimmune processes in real life. In the NOD mice the technique revealed that activation of naïve autoreactive T cells might occur in draining lymph nodes of the target organ. Furthermore, these studies indicated how regulatory mechanisms in vivo might control misled T cell responses. In EAE we learnt how fast the immune-privileged CNS converts to an immune permissive tissue, allowing millions of immune cells including autoaggressive T cells to

migrate through the tissue. It is well conceivable that this locomotion behavior might be harmful for the organ. Penetration of the compact tissue by the T cells may break up organized structures either via mere mechanical force and/or enzymatic digestion, and may directly affect interneuronal communication resulting in neurological deficiency. Furthermore, these studies showed T cells in contact with putative APCs throughout the parenchyma. Each of these contacts may trigger profuse secretion of cytokines, chemokines and other soluble mediators. Infusion of intravenous soluble brain antigen enhanced T cell activation within the CNS and aggravated clinical disease. This is a surprising observation, since APCs within the inflamed CNS should be permanently fed with myelin and other local autoantigens. Obviously, local autoantigen is not available at saturating concentrations. Suboptimal supply of autoantigen in the tissue may be responsible for the relatively low efficiency of naturally loaded APCs to present ex-vivo endogenous myelin protein. A direct T cell mediated damage to neurons was recently implicated in a study which followed the infiltration of freshly activated cultured autoreactive T cells into brain slices (Nitsch et al., 2004a). The T cells evoked Ca2+ fluxes when they came into contact with neurons. Obviously, many questions about the pathogenesis of CNS autoimmunity remain to be answered. TPM technology will most likely contribute to solve some of these. Further improvements can be expected on the side of the microscope technology as well as on the side of the marker design. Miniaturized (Helmchen et al., 2001) and flexible instruments (Bird and Gu, 2003) might help in gaining access to organs and to prolong the observation time. Better optics can be expected which increase the imaging depth and resolution (Helmchen and Denk, 2002b; Levene et al., 2004). Marker-dyes will enable a simultaneous observation of several distinct cell populations. The panel of transgenic animals carrying cell type specific marker-genes will expand, and functional markers will be developed, which allow a minute evaluation of cell functions in their natural milieu (Griesbeck, 2004; Xu et al., 2001; Mills et al., 2003; Wallrabe et al., 2003; Hadjantonakis et al., 2003). Finally, new animal models, e.g. spontaneous EAE models (Krishnamoorthy et al., 2006; Bettelli et al., 2006a) will allow to investigate new aspects of T cell mediated autoimmune diseases of the CNS. Acknowledgements The authors want to thank Sabine Kosin and Ingeborg Haarmann for excellent technical assistance. We are grateful to Dr Hartmut Wekerle for critical reading. This work was supported by the Deutsche Forschungsgemeinschaft (SFB455) and the Gemeinnützige Hertie foundation (grant no. 1.01.1/04/ 010 and 1.01.1/07/005). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2007. 09.017.

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