Djadja Jean

Delest Djadja

Ludovic Hamon

Sébastien George

Modeling and Evaluating of Human 3d+t Activities in Virtual Environment

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Modeling and Evaluating of Human 3d+t Activities in Virtual Environment

Djadja Jean Delest Djadja (B) , Ludovic Hamon (B) , and Sébastien George (B) LIUM -EA 4023, Le Mans Université, 72085 Le Mans, Cedex 9, France {djddja,ludovic.hamon,sebastien.george}@univ-lemans.fr

Abstract. This paper studies the problem of evaluation of human 3d+t activities in Virtual Environments (VE) for Learning (VEL). Current evaluation methods focus mostly on: (i) the automatic identification of an ordered sequence of actions and/or (ii), an empirical analysis made by experts through the VE. In many cases, the learner's activity can be represented by some specific time series made of geometrical data of 3D artefacts. For the extraction and analysis of such Motions Of Interest (MOI), one can manually segment them among the traces, and/or use automatic approaches requiring a database of annotated examples.

Both cases usually require too many resources to design such environments. Consequently, this work presents a method allowing teachers to quickly build, compare and evaluate a 3d+t learning activity in VE. This method is based on a semi-automatic approach combining the Dynamic Time Warping algorithm, with 3D reference shapes and few expert's demonstrations of the task to learn.

Keywords: Virtual reality Virtual Environments (VE) for learning (VEL) offer advanced interaction possibilities such as human movements and gestures for 3D objects selection, manipulation and navigation [START_REF] Emma-Ogbangwo | Enhancing user immersion and virtual presence in interactive multiuser virtual environments through the development and integration of a gesture-centric natural user interface developed from existing virtual reality technologies[END_REF][START_REF] Penichet | New Trends in Interaction, Virtual Title Reality and Modeling[END_REF]. Regarding the human activity, most of the time, it is evaluated after the activity execution in VEL [START_REF] Lee | Can a virtual reality surgical simulation training provide a self-driven and mentor-free skills learning? Investigation of the practical influence of the performance metrics from the virtual reality robotic surgery simulator on the skill learning and associated cognitive workloads[END_REF], according to an empirical evaluation and/or the use of task-dependant metrics characterizing the activity at the action level (done or not), and/or considering the search of an ordered sequence of actions. However, in many cases, it is crucial to characterize and evaluate each human action in terms of interactions and 3d+t features to provide an appropriate and real-time feedback in VEL [START_REF] Morel | Automatic and generic evaluation of spatial and temporal errors in sport motions[END_REF]. Although there are a lot of 3d+t metrics [3] to characterize a motion (velocity, jerk, curvature, etc.). They can have a pedagogical value only if they are related to a domain and a task-dependent analysis of the learning situation with some experts [START_REF] Toussaint | Towards using similarity measure for automatic detection of significant behaviors from continuous data[END_REF]. VEL based on this approach, require a heavy re-engineering process if the task or the application domain change. Usually, the re-engineering process cannot be made by the expert or the teacher [START_REF] Buche | MASCARET: a pedagogical multi-agent system for virtual environment for training[END_REF]. Furthermore, the identification of the Motions Of Interest (MOI) that would be analyzed, is a difficult task if we consider a VE where the user can start, pause and resume the learning scenario at anytime. Therefore, given a predefined task to learn, our method consists in automatically capturing, segmenting and evaluating targeted MOIs during the learning activity in VE. These MOIs represents the evolution in time and space (3d+t) of some monitored virtual artefacts resulting from the learner's activity in VE.

Our approach relies on the comparison between the expert performing the task and the learner thanks to some reference shapes considered as checkpoints, the Dynamic Time Warping (DTW) algorithm and some kinematic metrics [START_REF] Morel | Automatic and generic evaluation of spatial and temporal errors in sport motions[END_REF]. The details of our method and the implemented system are presented in the next section.

2 Activity Modeling and Evaluation: System Functioning and Use Cases Suppose a toy problem like a navigation task, where the learner has to walk according to two specific paths for reeducation purposes (Fig. 1, left). To set up the system, the expert (i.e. the expert of the task to learn) has to firstly select the virtual element to monitor (e.g. the learner's body). Next, the expert places three kinds of 3D reference shapes, acting as CheckPoints (CP) with which the artefact must collide with: a unique Starting and a unique Ending CP (SCP and ECP) for the task beginning and ending, and some optional Intermediate CPs (ICP). The ICPs represent sub-sequences of the task to learn. In case of several intermediate ICPs, the artefact must collide with them according to a sequential order. For the navigation example, boxes are used as CPs and the expert makes several demonstrations of a walking made of a curving path followed by a turning path. The SCP acts as an oriented local landmark from which, the positions and orientations of the artefact are computed and recorded until the ECP is reached. Finally, the teacher chooses one demonstration as a task to imitate. The learner tries to reproduce the task. The system will then record the learner's MOI from the SCP to the ECP. The shape of the learner's path will not necessarily match with the expert's demonstration. Therefore, the system compares the learner's MOI to the expert one by using DTW that gives a value indicating the similarity of two signals in terms of shape regardless of the duration (the lower the value is, the closer the two signals are, [START_REF] Morel | Automatic and generic evaluation of spatial and temporal errors in sport motions[END_REF]). If the value is acceptable (under a threshold), the learner can compare the performance to the expert's one thanks to a visual feedback, on a wall within the VE, that can display: the value of the DTW, the values of two kinematic metrics (for this example) the velocity and the jerk i.e. the rate of change of the movement acceleration. The lower the value is, the smoother the motion is [3] (Fig. 2, right). A replay of the learner's performance and/or the expert's demonstration is also available. Finally, the system functioning, with one ICP, is represented by the finite-state machine with the following transitions (Fig. 1,right): -a: SCP collision, go to state S i.e. the artefact evolution is monitored and recorded, the previous recording is deleted if existing -b: ICP collision, go to state I i.e. the ICP collides -c: ICP collision, go to state F, i.e. the expert's demonstration or the learner task fails -d (for the expert's demonstration): ECP collision, go to the ending state M, i.e. the recording stops, the metrics are computed and displayed within the VE. -e (for the learner evaluation): ECP collision, go to state D, i.e. the recording stops, the learner's performance is compared to the expert's one thanks to DTW -f: the DTW value is under or equal to the threshold, go to the ending state M -g: the DTW value is above the threshold go to the ending state F Two other simple toy problems related to (a) a throwing task of a ball and a manipulation task of a glass has been implemented (Fig. 2, demonstrations video can be found by following the link below1 ). The task (a) was built to address, in the future, an example of a throwing task. The monitored artefact is the ball that must be thrown in a bin according to a basketball launch (hand close to the head on start and elbow down, Fig. 2, left). The SCP is placed close to the user's head on her/his right (if right-handed) and attached to her/him to allow the launch of the ball around the bin. The task (b) illustrates the manipulation of object such a beaker in chemistry. This use case consists in monitoring a glass with a ball inside. The user must take the glass and put the ball into another container. The user must finish the task by turning over the glass and put it on the deposit area (Fig. 2, middle). In both tasks, the motions of the object are compared to the demonstration, thanks to DTW.

Conclusion and Future Work

With the proposed method, we hope that experts and teachers will be able to easily build a learning situation in VE and evaluate the learner's 3d+t activities. Once the checkpoints are placed in the scene and the demonstration is performed, the system can automatically record and analyse every learner's MOI, colliding an ordered sequence of virtual checkpoints and fitting the expert's demonstration. The future works will focus on the evolution of this prototype with the implementation of an appropriate interaction paradigm to allow teachers to build their own learning situations in VE. The limits and advantages of our method will also be studied by conducting an experiment to evaluate the authoring aspects of the proposed solution. The dart game and some simple chemistry exercises will be simulated to allow experts to put in place learning situations. The goal is to study the operationalization capacity of our system regarding the proposed learning scenarios. The learner evaluation process based on DTW and some kinematic metrics must be improved on three main points: (a) an inclusion of temporal aspects as the DTW algorithm works regardless of the signal duration, (b) a contextual and application-domain-dependent model to choose a minimal set of appropriate metrics to display within the VE and (c) visual feedbacks with affordance properties.

Fig. 1 .

 1 Fig. 1. (Left) Navigation task (Bird's eye); (Right) State machine for the expert's demonstration and learner evaluation with one ICP

Fig. 2 .

 2 Fig. 2. Left: throwing task (Side view); Middle: manipulation task of glass (front view); Right: Metrics display within the VE

•

  Activity evaluation • Motion •

	Human learning
	1Human3d+tActivityinVirtualEnvironment

https://www.dropbox.com/s/5t6e80j3oe85eiy/EC TEL

Activity 3dt.zip? dl=1.


Amelia Carolina Sparavigna

Change Detection in Satellite Images Applied to the Study of Sand Dunes

Keywords: Image Processing, Change Detection, Satellite Imagery, Climate Change, Sand Dunes

In recent years the research on Change Detection, applied to data and images obtained by remote sensing from satellites, has collected a huge number of publications, as we can easily see using Google Scholar or Semantic Scholar engines. For this reason, any review of this large research area must be necessarily fragmented according to specific applications. Here we consider the study of the motion of sand dunes.

The sets of time series of satellite images and data maps, which are reporting information collected by means of remote sensing technologies, are today the main datasets used in the research field of Change Detection. These sets of images and maps allow researchers to examine a given area over some specific periods of time, and determine the changes inside and investigate the related causes at the origin of variations. Besides monitoring the local environments, the trends extrapolated by the Change Detection can be used to forecast the evolution of the environmental conditions too. Not surprisingly therefore, we find, among the main subjects of the Change Detection, the analyses of deforestation, polar ice loss and all the global and local consequences of the Climate Change.

Several space agencies are contributing to provide time series of satellite images, useful for the previously mentioned problems. Images recorded on different dates can be statically compared or merged in a sequence, to obtain a multi-temporal image, by means of which we can easily observe the local changes. One of these agencies is ESA, the European Space Agency, which is running the Earth Watching Project. NASA, the National Aeronautics and Space Administration of the United States Federal Government, has service ARSET, Applied Remote Sensing Training, which offers a specific training to integrate the NASA Earth Science data into decision-making activities. Google, the American multinational technology company specialized in Internet-related products, has its service too. It is the Google Earth Timelapse, based on the time series of images in Google Earth.

The methods that we can use for a study of Change Detection can be roughly categorized into the two sets of supervised and unsupervised methods. The former set is based on supervised classification methods, which require additional information to obtain the desired features contained in the images or maps. The latter set performs change detection by means of a direct comparison of them, without incorporating any additional information [START_REF] Celik | Unsupervised change detection in satellite images using principal component analysis and k-means clustering[END_REF]. As explained in [START_REF] Celik | Unsupervised change detection in satellite images using principal component analysis and k-means clustering[END_REF], Change Detection by means of unsupervised methods uses the automatic analysis of change data, which are generally created as follow. First we have an image (or map) differencing. Then, this processing is followed by normalization and vector analyses [START_REF] Pacifici | An innovative neural-net method to detect temporal changes in high-resolution optical satellite imagery[END_REF]. Let us remember that image differencing-based algorithms accomplish the change detection by subtracting, on a pixel basis, the images acquired at two time instants to produce a new image called "difference image" [START_REF] Celik | Unsupervised change detection in satellite images using principal component analysis and k-means clustering[END_REF]. The changes need to be identified in the difference image. Some methods to detect the changes in images, referenced in [START_REF] Celik | Unsupervised change detection in satellite images using principal component analysis and k-means clustering[END_REF], are those given in [START_REF] Bruzzone | Automatic analysis of the difference image for unsupervised change detection[END_REF][START_REF] Boucher | A novel method for mapping land cover changes: Incorporating time and space with geostatistics[END_REF][START_REF] Gamba | Change detection of multitemporal SAR data in urban areas combining feature-based and pixel-based techniques[END_REF]. Some recent works are given in [START_REF] Leichtle | Unsupervised change detection in VHR remote sensing imagery-an object-based clustering approach in a dynamic urban environment[END_REF][START_REF] Cao | Unsupervised change detection in high spatial resolution remote sensing images based on a conditional random field model[END_REF][START_REF] Wu | Unsupervised object-based change detection via a Weibull mixture model-based binarization for high-resolution remote sensing images[END_REF][START_REF] Lv | Unsupervised change detection based on hybrid conditional random field model for high spatial resolution remote sensing imagery[END_REF][START_REF] Shao | Novel approach to unsupervised change detection based on a robust semi-supervised FCM clustering algorithm[END_REF][START_REF] Moghimi | Integrating thresholding with level set method for unsupervised change detection in multitemporal SAR images[END_REF][START_REF] Atasever | A new artificial intelligence optimization method for PCA based unsupervised change detection of remote sensing image data[END_REF][START_REF] Touati | Multimodal Change Detection in Remote Sensing Images Using an Unsupervised Pixel Pairwise Based Markov Random Field Model[END_REF][START_REF] Dharani | Land use and land cover change detection by using principal component analysis and morphological operations in remote sensing applications[END_REF].

In recent years, the research on the Change Detection collected a huge number of publications, as we can easily see using Google Scholar or Semantic Scholar engines. For this reason, any review of this large research area must be necessarily fragmented according to specific applications. Here we consider the study of the motion of sand dunes.

The sand dunes are a subject which has attracted many studies from the first pioneering works made by Ralph Alger Bagnold [START_REF] Bagnold | The Physics of Blown Sand and Desert Dunes[END_REF]. Today, instead of local explorations as those performed by Bagnold, we can easily study the dunes by means of the time series of satellite images. We can obtain the size distribution of the dunes, their motion, collision, and evolution (see for instance, [START_REF] Durán | The dune size distribution and scaling relations of barchan dune fields[END_REF][START_REF] Kumar | Assessment of sand dune change detection in Rajasthan (Thar) Desert, India[END_REF][START_REF] Necsoiu | Monitoring migration rates of an active subarctic dune field using optical imagery[END_REF]).

Actually, sand dunes can move and, during their motion, they can be a problems for human activities. Moreover, on the motion of sand dunes the climate change can strongly act, with disastrous consequences for local communities, as shown in [START_REF] Thomas | Remobilization of southern African desert dune systems by twenty-first century global warming[END_REF][START_REF] Redsteer | Monitoring and analysis of sand dune movement and growth on the Navajo Nation, southwestern United States[END_REF][START_REF] Boulghobra | Sand encroachment in the Saharan Algeria; the not declared disaster-Case study: In-Salah region in the Tidikelt[END_REF][START_REF] Boulghobra | Climatic data and satellite imagery for assessing the aeolian sand deposit and barchan migration, as a major risk sources in the region of In-Salah (Central Algerian Sahara)[END_REF].

The motion of the dunes has been studied by Change Detection in [START_REF] Sparavigna | A Study of Moving Sand Dunes by Means of Satellite Images (August 2, 2013)[END_REF][START_REF] Sparavigna | Moving dunes on the Google Earth[END_REF][START_REF] Sparavigna | The GNU Image Manipulation Program Applied to Study the Sand Dunes[END_REF][START_REF] Sparavigna | A Case Study of Moving Sand Dunes: The Barchans of the Kharga Oasis[END_REF]; the method of analysis is based on the difference of images (in [START_REF] Sparavigna | A Case Study of Moving Sand Dunes: The Barchans of the Kharga Oasis[END_REF], the dunes of the Kharga Oasis have been analysed; these dunes are also representing a problem for the archaeological remains of the Oasis [START_REF] Sparavigna | Remote Roman Fortifications: The Forts of the Kharga Oasis[END_REF]).

The method is the following [START_REF] Sparavigna | The GNU Image Manipulation Program Applied to Study the Sand Dunes[END_REF]. Two satellite images A and B of a dune field, recorded at different times and converted in grey tones, are merged in an image C. From image A, a new image A' is obtained by inverting the grey tones of it. Reducing its opacity to 50%, A' is copied as a layer on B. A' and B gives image C, an image like that shown in the Figure 1. In C, the dunes of image A appear as bright dunes, whereas the same dunes, recorded at a different time, and given by image B, appear in dark tones. We can join the two positions of the brink of each dune by a red line, to obtain the displacement of the dune. Since we have the dates when images have been recorded, we can easily obtain the speed of the dunes. Also the sedimentary patters of the dunes can be easily studied, by means of satellites [START_REF] Sparavigna | Sedimentary Patterns of Moving Sand Dunes in Orinoca District, Bolivia. Philica[END_REF][START_REF] Sparavigna | Analysis of the Motion of Some Brazilian Coastal Dunes[END_REF][START_REF] Tsoar | The effect of climate change on the mobility and stability of coastal sand dunes in Ceará State (NE Brazil)[END_REF] (see for instance the Figure 2). Moreover, the satellite images also show that the dunes can change their shapes, as it is happening for the barchans of the Laayoune-Sakia El Hamra region and Khenifiss National Park [START_REF] Sparavigna | Dunes changing their shape: The case of the dunes of the Laayoune-Sakia El Hamra region[END_REF][START_REF] Sparavigna | Sand Dunes of Khenifiss National Park of Morocco[END_REF] (see the Figure 3). Is this a consequence of the Climate Change? Figure 2: Sedimentary patterns of a dune field in Orinoca Canton, Bolivia. Note the "footprints" of the dunes (see [START_REF] Sparavigna | Sedimentary Patterns of Moving Sand Dunes in Orinoca District, Bolivia. Philica[END_REF]).

Figure 3: Barchans move and can change their shapes (the red circle is the reference point). Note the faint "ghost" shapes (marked in red) of the previous position and shape of a dune (more details in [START_REF] Sparavigna | Dunes changing their shape: The case of the dunes of the Laayoune-Sakia El Hamra region[END_REF][START_REF] Sparavigna | Sand Dunes of Khenifiss National Park of Morocco[END_REF]). The change in the shape was driven by a variation of the direction of prevailing winds. Is this variation induced by the Climate Change?

The Change Detection among images can be performed by many different approaches. In [START_REF] Varma | Change detection of desert sand dunes: a remote sensing approach[END_REF], the images are compared by means of a Radon Transform. Features based on the Radon spectrum are used to cluster dunes of various orientations. The dunes studied are those of the Gobi desert. A GIS-based method for extraction of sand sea encroachment was used for the dunes of the Rigboland Sand Sea, Central Iran [START_REF] Ahmady-Birgani | Sand dune encroachment and desertification processes of the Rigboland Sand Sea, Central Iran[END_REF].

According to the authors, their proposed analysis of the remote sensing images can be an effective tool for the monitoring and prognostication of sand dune movement and sand sea change.

Statistical parameters obtained by satellite images and wind data are used in [START_REF] Maghsoudi | The sand dunes migration patterns in Mesr Erg region using satellite imagery analysis and wind data[END_REF], for the dunes of the Mesr Erg region. In [START_REF] Els | Comparison of two Satellite Imaging Platforms for evaluating sand dune migration in the Ubari Sand Sea (Libyan Fazzan)[END_REF], the Ubari Sand Sea (Libyan Fazzan) is studied, by means of image overlay and differencing; the Root Mean Squared Error (RMSE) was used to determine if the mapped dune patterns were significantly different.

Of course, Change Detection is not only applied to satellite images. In [START_REF] Dech | Blowout dynamics on Lake Huron sand dunes: analysis of digital multispectral data from colour air photos[END_REF], a digital image analysis procedure was adapted to extract multispectral data from sequential colour air photos of a Lake Huron sand dune system. UAVs, Unmanned Aerial Vehicles, are also used [START_REF] Laporte-Fauret | Low-Cost UAV for high-resolution and large-scale coastal dune change monitoring using photogrammetry[END_REF][START_REF] Taddia | UAVs for structure-from-motion coastal monitoring: a case study to assess the evolution of embryo dunes over a two-year time frame in the Po River Delta, Italy[END_REF][START_REF] Pagán | Monitoring the dune-beach system of Guardamar del Segura (Spain) using UAV, SfM and GIS techniques[END_REF][START_REF] Grottoli | UAV application for monitoring the annual geomorphic evolution of a coastal dune in Punta Marina (Italy). Earth observation advancements in a changing world[END_REF]. In [42], by means of GIS techniques, it is the change of the elevation profile of coastal dunes, which is investigated. In fact, highly accurate and densely sampled coastal elevation data can be obtained by means of LIDAR topographic mapping (airborne Light Detection and Ranging). In [43], the coastal dunes of Cape Hatters have been investigated using this technology. In general, we can tell that the study of sand dunes and of other subjects of the Aeolian Science is today receiving a large benefit from the recent advances in Aerial and Information Technologies [START_REF] Scuderi | Application of database approaches to the study of Earth's aeolian environments: community needs and goals[END_REF].

To conclude, let us also note that the study of the sand dunes is not limited to Earth. Dunes are also present on Mars and they move [START_REF] Bourke | Recent aeolian dune change on Mars[END_REF][START_REF] Cardinale | Present-day aeolian activity in Herschel Crater[END_REF]. Then, the Change Detection can be applied also for these dunes too (Figure 4). Examples were proposed in [START_REF] Sparavigna | Sand Dunes Moving in the Nili Patera Caldera on Mars (August 2, 2013)[END_REF][START_REF] Sparavigna | Edge-Detection Applied to Moving Sand Dunes on Mars[END_REF]. NASA/JPL-Caltech/Univ. of Arizona/JHU-APL). See [START_REF] Sparavigna | The GNU Image Manipulation Program Applied to Study the Sand Dunes[END_REF] for more details.

Mars is a sandy planet. The HiRISE camera on the Mars Reconnaissance Orbiter (MRO) is the powerful tool which is giving us detailed pictures of Martian dunes. On Mars, strong and sustained winds have been considered rare. However, in the case of the Nili Patera dune field, the dunes have unexpectedly high sand fluxes, similar to those we can find in Victoria Valley, Antarctica [START_REF] Bridges | Earth-like sand fluxes on Mars[END_REF]. This fact induced the conclusion that rates of landscape modification on Mars can exist, similar to those observed on Earth.

Figure 1 :

 1 Figure 1: Dunes move, as we can see using the time series of Google Earth. See Ref.25 for more details.

Figure 4 :

 4 Figure 4: The red and blue pixels are showing the ripples on a dune in the Nili Patera Caldera on Mars, from two images recorded at different time by HiRISE, the High Resolution Imaging Science Experiment (Credit:NASA/JPL-Caltech/Univ. of Arizona/JHU-APL). See[START_REF] Sparavigna | The GNU Image Manipulation Program Applied to Study the Sand Dunes[END_REF] for more details.

Zenodo, 26 September 2019 -DOI 10.5281/zenodo.3461709


Kevin Pasini

Allou Same

Mostepha Khouadjia

Fabrice Ganansia

Latifa Oukhellou

Representation Learning of public transport data. Application to event detection

come

Representation Learning of public transport data. Application to event detection. Kevin Pasini* 1,2 , Allou Samé 1 , Mostepha Khouadjia 2 , Fabrice Ganansia 3 , Latifa Oukhellou 1 1 Université Paris-Est, French Institute of Science and Technology for Transport, Development and Network (IFSTTAR), 14-20 Bd Newton 77420 Champs-sur-Marne, France 2 IRT SystemX, Site du Moulon. Bât. 660 -Rue Noetzlin. 91191 Gif-Sur-Yvette, Saclay, France. 3 SNCF-Innovation & Recherche, Immeuble Le Jade, Bureau B2-18, 1-3 avenue François Mitterrand. 93210 La Plaine St Denis, France. * Corresponding author: kevin.pasini@irt-systemx.fr

Extended Abstract

A great deal of current research is focusing on mining large scale mobility data with exploratory, clustering or prediction purposes. Regarding public transport, numerous studies focus on prediction of travel demand [START_REF] Wang | Research on subway passenger flow combination prediction model based on rbf neural networks and lssvm[END_REF] [2] [3] [4] [START_REF] Heydenrijk-Ottens | Supervised learning: Predicting passenger load in public transport[END_REF]. This forecasting can be used for transportation planning when longterm time horizon is considered. It can also be used by public transport authorities and transport operators in the case of short/medium-term time horizon either to enrich public transport route planner or to help the co-regulation of the transport network.

On the basis of data collected by counting sensors deployed on trains, this paper deals with a forecasting of passenger load in public transport taking into account train operation. Providing passengers with train load forecasting, in addition to the expected arrival time of the next train, can indeed be useful for a better planning of their journeys, which can prevent over-crowding situations in the trains [START_REF] Ceapa | Avoiding the crowds: Understanding tube station congestion patterns from trip data[END_REF] [START_REF] Lathia | Mining mobility data to minimise travellers' spending on public transport[END_REF]. The proposed approach is built on both a hierarchy of recurrent neural networks [START_REF] Cho | Learning phrase representations using rnn encoder-decoder for statistical machine translation[END_REF] and representation learning [START_REF] Bengio | Representation learning: A review and new perspectives[END_REF] with the aim to explore the ability of such mobility data processing to simultaneously perform a forecasting task and highlight the impact of events on the public transport operation and demand. An event refers here to an unexpected passenger transport activity or to a modification in transport operation compared to those corresponding to normal conditions. Two kind of historical data are used, namely train load data and automatic vehicle location (AVL) data. This latter source contains all information related to the train operation (delay, time of arrival/departure of vehicles ...). The proposed methodology is applied on a railway transit network line operated by the French railway company SNCF in the suburban of Paris. The historical dataset used in the experiments covers the period from 2015 to 2016.

Two contributions will be highlighted in this paper:

-The first contribution is related to the passenger load forecasting. Most of the researches carried out in this domain deals with the issue of prediction at an aggregated level, where the time step is considered to be fixed. We focus here on the forecasting at non-aggregated level taking into account real time train schedules. This induces an irregular time step on the train load series to be predicted. The prediction method we propose is able to take into account contextual factors such as calendar information (day, time, holiday ...) and train operation. Combining iteratively recurrent neural networks with representation learning on contextual factors allows us to perform prediction of the train load. -The second contribution is to show how to explore the latent space provided by the neural network in order to highlight the impact of events on both the train operation and the train load. This new descriptive analysis is conducted by extracting from the initial latent space additional features according to which the data can easily be grouped into homogeneous clusters [START_REF] Jiang | Variational deep embedding: an unsupervised and generative approach to clustering[END_REF][START_REF] Yang | Towards k-means-friendly spaces: Simultaneous deep learning and clustering[END_REF].

Following this line of research, an embedding of the input data combined with a clustering approach are used to learn efficient representation of mobility data, which allows to reflect the impact of event. Such approach is able to learn representation for mobility data while clustering them. Indicators are then defined to qualify the impact of the event.

The experiments are carried out on real data collected on a railway line operated by the French railway company SNCF. This line serves the north area of suburban of Paris and carries around 250.000 daily passengers. As it can be seen in Figure 1, this line can be divided into four branches serving different terminals. This transport network structure induces a complex railway operation of this line. The dataset includes both counting data of passengers boarding and alighting at each station and the timetable information. These heterogeneous sources of data lead us to reconstruct the passenger load of each train per inter-stations as shown on Figure 1. An in-depth analysis of the obtained results will be detailed in the paper to show the ability of the proposed approach to outperform classical machine learning models in terms of multi-step prediction performances. Moreover, indicators defined on the latent space will be exhibited and analyzed to quantify their capacity to highlight the impact of the events.


C Vuaillat

M Varrin-Doyer

A Bernard

I Sagardoy

S Cavagna

I Chounlamountri

M Lafon

Pascale Giraudon
email: giraudon@lyon.inserm.fr

High CRMP2 expression in peripheral T lymphocytes is associated with recruitment to the brain during virus-induced neuroinflammation

Keywords: neuroimmunology, neurovirology, neuroinflammatory disease, T-cell motility, rabies virus, canine distemper virus

High CRMP2 expression in peripheral T lymphocytes is

associated with recruitment to the brain during virus-induced neuroinflammation.

1-Introduction

In acute viral encephalitis, the recruitment of immune cells to the central nervous system (CNS) plays a crucial role in the outcome of this disease. Notably, T-lymphocytes are key players in the initiation of specific intrathecal immune responses by directly destroying virus-infected cells, increasing the phagocytotic activity of macrophages and stimulating the production of antibodies by B cells [START_REF] Fujinami | Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease[END_REF][START_REF] Salazar-Mather | Cytokine and chemokine networks: pathways to antiviral defense[END_REF][START_REF] Tishon | CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B cells alone are ineffective[END_REF]. Thus, immune cells control the spread of viruses and participate in viral clearance. However, T-lymphocytes can mediate inappropriate inflammatory responses, producing multiple cytokines, chemokines, and reactive oxygen species that contribute to severe neural tissue injury and CNS dysfunction [START_REF] Carlson | The pathologic role for COX-2 in apoptotic oligodendrocytes in virus induced demyelinating disease: implications for multiple sclerosis[END_REF]) [START_REF] Peterson | Inflammation, demyelination, neurodegeneration and neuroprotection in the pathogenesis of multiple sclerosis[END_REF][START_REF] Galelli | Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis[END_REF][START_REF] Giraudon | Semaphorin CD100 from activated T lymphocytes induces process extension collapse in oligodendrocytes and death of immature neural cells[END_REF][START_REF] Lane | A central role for CD4(+) T cells and RANTES in virusinduced central nervous system inflammation and demyelination[END_REF][START_REF] Patterson | Measles virus infection induces chemokine synthesis by neurons[END_REF]. Considering the importance of viral encephalitis in humans, the homing behavior and functional status of immune cells that infiltrate the virus-infected brain need to be clarified.

Characterizing the mechanisms that favor the migration of T-lymphocytes to the CNS in response to viral infection is of interest both for understanding the physiopathology of virus-induced neuroinflammation and to propose new therapeutic approaches.

T-lymphocytes constantly move in and out of lymph nodes, circulating through the vasculature.

Probably following activation and as a result of chemotactic stimuli, T-cells acquire a polarized morphology, leave the vasculature and start to crawl within neural tissue [START_REF] Norman | Mechanisms of lymphocyte migration in autoimmune disease[END_REF][START_REF] Sanchez-Madrid | Leukocyte polarization in cell migration and immune interactions[END_REF][START_REF] Westermann | Naive, effector, and memory T lymphocytes efficiently scan dendritic cells in vivo: contact[END_REF]. Dynamic cytoskeletal remodeling is fundamental to drive the rapid conversion of lymphocyte cytoarchitecture from a semi-rigid to a highly deformable state needed for transendothelial migration and crawling within CNS parenchyma [START_REF] Vicente-Manzanares | Role of the cytoskeleton during leukocyte responses[END_REF]. Thus, motile T-cells display a polarized morphology, with two distinct T-cell compartments. At the advancing front, the leading edge is enriched in chemokine receptors and acts as a sensor to the microenvironment. At the trailing edge, the uropod is an adhesive and retractile structure required for T-cell migration [START_REF] Barreiro | Interactive protrusive structures during leukocyte adhesion and transendothelial migration[END_REF].

Rapid reorganization of actin at the leading edge and the retraction of microtubule and vimentin networks in the uropod during polarization and migration have been well described [START_REF] Brown | Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments[END_REF][START_REF] Mitchison | Actin-based cell motility and cell locomotion[END_REF]. Such a cytoskeletal reconfiguration requires a complex and still not completely defined array of intracellular signaling pathways implicating second messengers, several kinases and Rho family small GTPases [START_REF] Del Pozo | Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes[END_REF][START_REF] Vicente-Manzanares | A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis[END_REF]. In search of molecules involved in cell motility and shared by the immune and nervous systems, we recently identified Collapsin Response Mediator Protein-2 (CRMP2) in T-lymphocytes [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF], a phosphoprotein described in neuron growth cone advance (Goshima et al, 1995) and neural cell migration via microtubule organization. We showed that CRMP2 redistributes in the uropod in motile T-cells, binds cytoskeletal elements and is implicated in T-cell polarization and migration [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF]. A correlation between CRMP2 expression levels and cell migratory rates was demonstrated by over-expression and knock-down experiments in primary T-lymphocytes.

The importance of CRMP2 in the immune response was emphasized by the observation that, in humans suffering from virus-induced neuroinflammatory disease, peripheral activated immune cells displayed high migratory rates associated with elevated CRMP2 expression [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF].

These observations led us to suspect a role for CRMP2 in immune cell trafficking and recruitment to the inflamed CNS. In the present study, we evaluated the CRMP2 contribution in brain lymphocyte recruitment by analyzing CRMP2 expression in peripheral and brain-infiltrating immune cells in mouse models of infection using neurovirulent and non-neurovirulent strains of rabies virus (RABV) and Canine distemper virus (CDV). After inoculation of the neurovirulent RABV strain Challenge virus standard (CVS) in the muscle of the hind limb-to mimic the inoculation of this virus by an animal bitethe virus enters nerve endings via the neuromuscular junction and then travels from one neuron to another along the spinal cord before reaching the entire brain, causing severe neuronal dysfunctions and fatal encephalitis [START_REF] Lafon | Subversive neuroinvasive strategy of rabies virus[END_REF]. Infection in the CNS triggers a strong innate immune response in both neurons and astrocytes, resulting in inflammatory and chemo-attractive responses characterized by IFN, IL-1, IL6, TNF CCL5, CXCL-10 expression capable of attracting T cells, B cells and macrophages through the blood brain barrier (Camelo et al., 2001a;[START_REF] Baloul | Up-regulation of Fas ligand (FasL) in the central nervous system: a mechanism of immune evasion by rabies virus[END_REF][START_REF] Wang | Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system[END_REF][START_REF] Roy | Failure to open the blood-brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection[END_REF]. Inoculation of the Pasteur Virus (PV) RABV strain another neurovirulent RAV strain causes only spinal cord and the bulb infection, resulting in abortive rabies: mice cleared the CNS infection and survive despite paralytic sequel (6). By contrast, injection by the same route of the non-neuronotropic vaccine RABV strain ERA does not cause infection of the CNS and animals remain healthy. In the mouse model infected with CDV, intracerebral inoculation of a neurovirulent strain results in acute encephalitis followed, in surviving animals, by the development of motor impairment and metabolic disease [START_REF] Bernard | Alteration of the leptin network in late morbid obesity induced in mice by brain infection with canine distemper virus[END_REF][START_REF] Griffond | Specific alteration of the expression of selected hypothalamic neuropeptides during acute and late mouse brain infection using a morbillivirus: relevance to the late-onset obesity?[END_REF][START_REF] Verlaeten | Down regulation of melanin concentrating hormone in virally induced obesity[END_REF]. CDV infects the neurons of defined brain structures [START_REF] Bernard | Brain structures selectively targeted by canine distemper virus in a mouse model infection[END_REF] and provokes a brain inflammatory reaction with the infiltration of T lymphocytes, the expression of the cytokines TNF, IL6, IFN and IL-4 associated with the disruption of the tissue inhibitor of the metalloprotease TIMP/ metalloprotease MMP balance (Benscsik et al., 1996;[START_REF] Khuth | Morbillivirus infection of the mouse central nervous system induces region-specific upregulation of MMPs and TIMPs correlated to inflammatory cytokine expression[END_REF] capable of modifying vascular permeability.

We show here that, in contrast of what was observed in mice infected with non-neurovirulent virus strain, infection of the CNS with neuronotropic CDV and RABV enhanced both CRMP2 expression and migratory rate of activated T lymphocytes. Interestingly, the presence in blood and lymph nodes of lymphocytes expressing high CRMP2 level was associated with the intensity of recruitment of immune cells into the brain of mice suffering from encephalitis and correlated with the severity of clinical score.

2-Material and Methods

-Animal models

Mice, rabies infection and assessment of clinical symptoms

Experiments were performed with 6-week-old female BALB/c mice from Janvier (St. Berthevin, France). Mice were inoculated intramuscularly in both hind legs, with 1x10

Mice, CDV infection and clinical symptoms

Four-week-old female outbred SV129 mice (Harlan-France) were inoculated intracerebrally (10l) as already reported (Bernard et al., 1983)

-Cell preparation

For peripheral blood mononuclear cell (PBMC) analysis, blood was collected at the retro-orbital plexus. PBMCs were then isolated on Ficoll-Hypaque (Eurobio), washed three times with PBS and resuspended in PBS/2%FCS before immunocytochemistry and flow cytometry analysis. For lymph node mononuclear cell analysis, mononuclear cells were collected from popliteal (for RABV model) or cervical lymph nodes (for CDV model), following dissociation with a syringe plunger. Lymphocyte enrichment was performed by a 30 min contact of the cell preparation on a plastic dish to remove adherent phagocytes. Mononuclear cells infiltrating the CNS were collected using Percoll (Amersham Biosciences) gradients, as previously described (Camelo et al., 2001b).

-Flow cytometry phenotyping

Mononuclear cells were washed in PBS and then incubated for 15 min at room temperature (RT) with various combinations of fluorescence-conjugated mAbs (all from Beckman Coulter, unless otherwise specified) as follows: phycoerythrin (PE)-labeled anti-CD3, PE-labeled anti-CD69 and PE-labeled anti-CD11c. The stained cells were washed and fixed with 4% paraformaldehyde (PFA-Sigma) in PBS (pH 7.4) for 30 min at 4°C and washed again. Cells were then re-suspended in PBS at 4°C awaiting analysis (if surface staining alone was required) or processed further for intracellular CRMP2 protein detection. For the latter, cells were re-suspended in PBS containing 0.5% saponin (permeabilization buffer -PB) and incubated for 30 min at RT with rabbit anti-peptide4/CRMP2 antibody, prepared against a CRMP2 [START_REF] Ricard | Isolation and expression pattern of human Unc-33-like phosphoprotein 6/collapsin response mediator protein 5 (Ulip6/CRMP5): coexistence with Ulip2/CRMP2 in Sema3a-sensitive oligodendrocytes[END_REF]. The cells were washed once with PBS 0.05% saponin and then incubated in PBS 0.5% saponin with fluorescein isothiocyanate (FITC)-labeled goat F(ab')2 antirabbit for 20 min at RT. Finally, the cells were washed with PBS 0.05% saponin, re-suspended in PBS, and analysed by flow cytometry on a Coulter EPICS XL (Beckman Coulter). Nonspecific staining was excluded by isotypic control gating.

-Immunohistochemistry

Analysis of infiltrating T cells was performed on brain slices using two different protocols of fixation. 1)

For Diaminobenzidine staining, mice were perfused with PBS then PBS containing 1% PFA. Removed brains were then post-fixed in 1% PFA (1h, RT), immersed in 15% sucrose (overnight), frozen in isopentane cooled to -60°C and stored at -80°C until use. Coronal brain sections (cryo microtome, 14 to 20m thick) from mice sacrificed on day 14 post-infection with CDV were collected on Superfrost glass slides. Following treatment with reagent to eliminate nonspecific labeling due to endogenous biotin (Vector kit), slides were incubated for 24h with anti-mouse CD4 monoclonal antibody (clone H129.19, Pharmingen Int.). Samples were then sequentially washed and incubated with biotinylated anti-rabbit or anti-mice IgG antibodies (2h, RT), avidin-biotin peroxidase complex (VectaStain, 1h RT) and 3.3'-diaminobenzidine. 2) For immunofluorescence analysis, brain were removed and immediately frozen in isopentane cooled to -60°C and stored at -80°C until coronal sections and use. Fixation in absolute ethanol (10 min, RT) was required for CD4 and CD8 detection while acetone fixation (10 min, 4°C) was required for CRMP2 and TCR beta chain detection. Following 1h incubation with a blocking solution (PBS, 0,3% Triton, 1% BSA, 1% normal goat serum) to eliminate nonspecific labeling, slides were incubated with anti-mouse CD4 and CD8 (clone H129.19 and clone53-6.7, Pharmingen Int.) monoclonal antibodies or polyclonal anti-peptide4/CRMP2 antibody [START_REF] Ricard | Isolation and expression pattern of human Unc-33-like phosphoprotein 6/collapsin response mediator protein 5 (Ulip6/CRMP5): coexistence with Ulip2/CRMP2 in Sema3a-sensitive oligodendrocytes[END_REF]. (2h, 37°C), then with anti-rat and anti-rabbit IgG Alexa 488 antibodies (2h, RT) (Molecular Probes, Inc.). T-cell receptor was detected using a FITC anti TCR beta chain (clone H57-597, BD Pharmingen). Omission of the primary or secondary antibody resulted in no signal.

-Transmigration assay

Transmigration of lymphocytes, isolated from lymph nodes as reported above, was performed both in micro-Transwell system as described [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF] and in organotypic culture of mice brain.

For ex vivo transmigration on mice CNS, hippocampus cultures were prepared as follows. Hippocampi from postnatal (P7) C57BL6 mouse were dissected and placed immediately in cold Gey's balanced solution supplement with glucose (6,5mg/ml). Four hundred micrometer slices were cut perpendicularly to the septotemporal axis of the hippocampus using a McIllwain tissue chopper. Slices were carefully trimmed for excess tissue, and 6 slices were placed immediately on 30 mm semipermeable membrane inserts (Millicell-CM, Millipore) in a 6-well plate containing 1 ml of culture medium in each well. The culture medium consisted of 50% Minimum Essential Medium (Gibco), 25%

Hank's balanced salt solution, 25% heat-inactived horse serum (Gibco), 1% L-glutamine 200Mm

(Gibco) and 6,5 mg/ml D-glucose. Plates were incubated at 37°C and 5% CO2. The culture medium was exchanged twice a week. Lymphocytes (1.10 6 cells per slice) stained ex vivo using the vital fluorochrome carboxyfluoroscein succinimidyl ester/CFSE (1mM, 5 min, 37°C) were spotted closely to hippocampus slices. Following a 18h contact at 37°C, slices were extensively washed with D-MEM, fixed with ethanol (10 min, 4°C), incubated with 4', 6'-diamidino2-phenylindol/DAPI for nucleus staining. Numbers of infiltrating lymphocytes were counted under fluorescence microscopy.

For ex vivo transmigration in micro-Transwell system (Boyden chamber, Costar, 3 m-diameter pore size membrane coated with fetal calf serum, in triplicate), lymphocyte preparations (4.10 5 cells/well) were added in the upper chambers and chemokines in the lower compartments: CXCL12 (10ng/ml), CCL5 (100ng/ml), CCL2 and CXCL10 (20ng/ml). Transwell were incubated at 37°C for 1.5-2h. In blockade experiments, anti-pep4 antibody CRMP2 blocking antibody (3 mg/mL) was added to the lymphocyte preparation for 30 min at 37°C just before transmigration assay as already reported [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF]. Migratory cells in the lower chambers or in hippocampus slices were counted under the microscope. Data were expressed as total number of migratory T cells.

Statistical analysis

Statistical significance in comparing two means was tested with the unpaired Student's t test, p values<0.05 were considered significant. In the migration test, the number of migratory lymphocytes was counted by light microscopy (15-20 microscope fields per condition -2 or 3 independent experiments) and data expressed as mean number of migratory lymphocytes per field.

3-Results

Neuronotropic RABV and CDV infections trigger the stimulation of CRMP2+ cell populations in the periphery

Mouse models of virus-induced encephalitis, RABV and CDV were developed using two different strains of mice, C57B6 and SV129. Preliminary analysis indicated that the basal expression of CRMP2 BALB/C mice were infected with three different types of laboratory RABV strains: CVS, PV and ERA.

These strains are different in respect of neurotropism and issue of the disease. ERA is a vaccine virus strain, which has lost the property to infect the nervous system after peripheral infection in BALB/c mice. PV is a neuronotropic virus causing only abortive rabies [START_REF] Galelli | Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis[END_REF] as mice are paralyzed but survived. In contrast, CVS is a neuronotropic virus strain causing a fatal myeloencephalitis [START_REF] Baloul | Up-regulation of Fas ligand (FasL) in the central nervous system: a mechanism of immune evasion by rabies virus[END_REF]. PBMC and lymph nodes were sampled at different times (days 1, 7 and 9) after intramuscular injection with RABV or injection of medium only (non-infected mice). After CVS inoculation, infection did not significantly modify the % of CD3 + cells among the PBMC of BALB/c mice during the time period (days 1, 7 and 9) of the infection (Figure 1-A). In contrast, the percentage of cells expressing CD69, this molecule being taken as a marker of early activation [START_REF] Hamann | Expression cloning of the early activation antigen CD69, a type II integral membrane protein with a C-type lectin domain[END_REF], increased significantly on day 7 before declining thereafter, suggesting that infection with CVS triggered the transient activation of PBMC (Figure 1-A). In parallel, a drastic increase in CRMP2 + cells among the PBMC of CVS-infected animals occurred at day 7 (78% of the PBMC in infected animals expressed CRMP2 compared to less than 20% in non-infected animals) (Figure 1A). This increase was still high (70%) on the 9 th day of infection. The CRMP2+ cells recruited among the CD3+ T lymphocytes and activated CD69+ cell subsets, as shown by double-staining analysis (80% of CRMP2+ cells from CVS infected animals at day 7 were CD3+ and 20% were CD69+) (Figure 1-B).

Virtually all CD69+ cells (98.7±1.2%) triggered by the infection at day 7 and most CD3+ T cells (75.4±2.8%) expressed CRMP2. In addition, the frequency of T lymphocytes expressing CRMP2 increased with time (20% on day 1, 70-80% on days 7 and 9, respectively (Figure 1-C). Similar patterns were obtained with popliteal lymph nodes (data not shown). After infection with PV, a neuronotropic RABV strain causing abortive rabies, similar expansion of CRMP2+ cells was observed in PBMC than those observed after CVS infection (Figure 1D). In contrast, after infection with the nonneuronotropic RABV strain ERA, the number of cells expressing CRMP2 remained unchanged in the course of the infection and not different from those observed in non-infected mice. However, expansion of CD69 positive cells was similar in ERA to those observed in PV infected mice, suggesting that the early steps of T cell activation have occurred after ERA infection as they have occurred after PV and CVS infection. This suggests that CRMP2 expression by lymphocytes in periphery is associated with RABV neurotropism.

Altogether, these data indicated that neurotropic strains of RABV triggered CRMP2 expression in peripheral T lymphocytes and that most of the early-activated cells (CD69+), which increase in number through infection, also expressed CRMP2.

CRMP2 expression was then evaluated in a model of mice infected by intracerebral injection of a mice-adapted neurovirulent CDV strain previously shown to infect neurons [START_REF] Bernard | Brain structures selectively targeted by canine distemper virus in a mouse model infection[END_REF]. In Altogether, these observations supported the idea that infection of the CNS with a neurotropic virus modifies CRMP2 expression in activated immune cells in the periphery, and favored the recruitment, mainly of CRMP2+ T-lymphocytes, to the infected brain.

Enhanced migratory rates for peripheral immune cells displaying elevated CRMP2 expression in CDV-infected mice

As brain infiltration may result from the elevated migratory rate and/or enhanced survival of activated immune cells within neural tissue, the effect of CNS viral infection on the motility of peripheral immune cells was investigated ex vivo on T-lymphocytes isolated from lymph nodes on day 7 post inoculation

(3 infected-and 3 non-infected mice tested). Immune cells from lymph nodes were first analyzed for CRMP2 expression. Cytofluorimetry detected a clear increase in CRMP2 expression level (Figure 6-A)

and the presence of CRMP2hi cells (38.3 to 77.2% of the total CD69+CRMP2+ cells) in CDV-infected mice. The migratory rate was further analyzed, as previously reported [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF], in a

Transwell chamber system allowing the determination of the number of cells migrating towards a gradient of chemokines known to be present in virus-infected inflamed brain (CXCL12, CCL5, CXCL10) [START_REF] Patterson | Measles virus infection induces chemokine synthesis by neurons[END_REF]. As shown in Figure 6-B, lymphocytes from CDV-infected mice displayed a greater migratory rate compared to those isolated from non-infected mice (89,1±4 versus 43,7±17 migrating cells). In blockade experiment using an anti-CRMP2 antibody previously shown to lower T cell migration, the migratory rate was reduced by 30% and 10% for lymphocytes of CDV-and sham-inoculated mice, respectively. The ability for lymphocyte to migrate to CNS tissue was further examined ex vivo using mouse hippocampus maintained in organotypic culture (Figure 6-C). Brain slices were either non-infected or infected with CDV for three days before migration assay.

Lymphocytes from lymph nodes of CDV-infected and sham-inoculated mice were stained with vital dye CFSE (1M) then spotted close to CDV-and non-infected brain slices. Following 18h incubation, migrating lymphocytes were detected on brain slices (Figure 6-C). Compared to lymphocytes of shaminoculated mice, lymphocytes of CDV-infected mice greatly invaded the CDV-infected brain slices (Figure 6-C and graph in 6-D). Following treatment with anti-CRMP2 antibody, lymphocytes of CDVand sham-inoculated mice displayed similar migratory rates, demonstrating the role of CRMP2 in the elevated ability to traffic to neural tissue displayed by CDV-infected mice lymphocytes.

All these experiments indicated that virus-induced elevated CRMP2 expression conferred to lymphocytes a greater ability to migrate into neural tissue, thus increasing the potential of T cells to target infected/inflamed brain.

Presence of peripheral CRMP2hi cells correlates with the severity of clinical scores in mice suffering from RABV encephalitis

In contrast to mice infected with CDV, which develop abrupt signs of encephalitis, RABV-infected mice displayed progressive and well-scored clinical signs (Camelo et al., 2001b), allowing a correlative study between clinical score and CRMP2 expression levels in the periphery. After intra-muscular injection with the highly neurotropic strain of RABV, CVS-1, BALB/c mice develop fatal acute encephalitis. The first symptoms (ruffled fur, muscle stiffness) detected at day 6 post-infection (pi)

were quickly followed by paralysis of the hind limbs and then forelimbs, hunch back, severe loss of weight and prostration followed by death (day 13 pi). A representative evolution of the cumulative scores (addition of individual scores of the 8 animals of the group) after RABV injection is presented in causing abortive RABV and sustained infiltration of T cells into the CNS [START_REF] Galelli | Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis[END_REF], the correlation index was 0,78. This strongly suggested the participation of CRMP2hi cells in the deleterious neuroinflammatory reaction induced in the infected CNS, and indicated CRMP2 expression levels in PBMC as a potential peripheral marker of virus-induced neuroinflammation.

Collectively, our data revealed an association between clinical features and the presence, both in the periphery and in the brain of mice suffering from virus-induced encephalitis, of activated Tlymphocytes displaying high CRMP2 levels.

4-Discussion

In the present study, we showed that brain infection by neurovirulent strains of two viruses, either RABV or CDV, causing fatal encephalitis, triggers CRMP2 expression in activated T lymphocytes.

These cells of increased mobility find their way to the infected brain. CRMP2 expression by T cells seems to be a hallmark of infection/inflammation of the brain, since it was not observed after infection with non-neurovirulent RABV infection. Interestingly, the kinetics of CRMP2 expression in the periphery reflected the severity of the brain disease.

Following intramuscular (IM) inoculation, neurovirulent strains of RABV induced local (popliteal lymph node) and systemic (spleen) proliferative and cytotoxic responses, with notably cytokine secreting T cells and specific RABV antibodies [START_REF] Irwin | Basis of rabies virus neurovirulence in mice: expression of major histocompatibility complex class I and class II mRNAs[END_REF]. We showed here that, as early as 5 days post infection, the T cells of lymph nodes and blood were activated (CD69 expression) and expressed CRMP2. After intracerebral (IC) injection of CDV, a similar immune response was obtained,

suggesting that viral inoculation by the IM and IC routes both activated the systemic immune response. This can be surprising as the nervous system is an immuno specialized organ where directly injected viruses evade an immune response, probably through the installation of an immunosuppressive milieu [START_REF] Galea | What is immune privilege (not)?[END_REF]. However, this is not the case when antigens or virus particles are delivered by intrathecal immunization (Cserr et al., 1992;Cserr and Knopf, 1992;[START_REF] Harling-Berg | Myelin basic protein infused into cerebrospinal fluid suppresses experimental autoimmune encephalomyelitis[END_REF][START_REF] Panda | Immunologic Response to Subarachnoid and Intracerebral Injection of Antigens[END_REF][START_REF] Stevenson | Recruitment, activation and proliferation of CD8+ memory T cells in an immunoprivileged site[END_REF][START_REF] Shin | Efficacy of intracerebral immunization against pseudorabies virus in mice[END_REF]. It is likely that after IC inoculation of the CDV, virus induced leakage of neural cells and redirected viral antigens to the cerebrospinal fluid, the meninges and finally to the cervical lymph nodes [START_REF] Hatterer | How to drain without lymphatics? Dendritic cells migrate from the cerebrospinal fluid to the B-cell follicles of cervical lymph nodes[END_REF].

Thus, despite the tropism of neurovirulent RABV and CDV to the CNS, initial viral entry into the body by the IM and IC routes triggered a strong immune response in the periphery and the induction of CRMP2 positive cells in secondary lymphoid organs that circulate further into the blood. After IM infection with a non-neurovirulent RABV strain, CRMP2 expansion was no observed. This does not result from an absence of immune activation since expansion of CD69+ cells was similar to those observed after neurovirulent RABV injection, nor from the route of injection since both groups of mice were injected IM. It may result of the capacity of the non neurovirulent RABV strain ERA to infect bone marrow macrophages [START_REF] Ray | Lodmell Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence[END_REF] a property which is not shared by neurovirulent RABV strains which are strictly neuronotropic. This point would require further investigation.

How could CRMP2 integrate the multiple pathways involved in the homing and accumulation of T lymphocytes in the infected CNS? Access to the brain is strictly controlled by blood-brain interfaces, blood vessels and the choroid plexus, and accumulation in neural tissue may result from the enhanced entry/reduced exit rates of T cells from the brain [START_REF] Kleine | Immune surveillance of the human central nervous system (CNS): different migration pathways of immune cells through the blood-brain barrier and bloodcerebrospinal fluid barrier in healthy persons[END_REF][START_REF] Bechmann | What is the blood-brain barrier (not)?[END_REF][START_REF] Engelhardt | The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms[END_REF]. The activation process has a dramatic effect on this pathway, in particular by modulating the expression of the adhesion molecules, metalloproteases and chemokine receptors required for lymphocyte transendothelial migration and crawling in inflamed tissue [START_REF] Barreiro | Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes[END_REF][START_REF] Del Pozo | Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway[END_REF][START_REF] Giraudon | Cytokines secreted by glial cells infected with HTLV-I modulate the expression of matrix metalloproteinases (MMPs) and their natural inhibitor (TIMPs): possible involvement in neurodegenerative processes[END_REF][START_REF] Lane | A central role for CD4(+) T cells and RANTES in virusinduced central nervous system inflammation and demyelination[END_REF][START_REF] Strazielle | Pro-inflammatory cytokines modulate matrix metalloproteinase secretion and organic anion transport at the blood-cerebrospinal fluid barrier[END_REF]. As a result, more resting T-cells are found in lymphoid organs while activated T-cells mainly target nonlymphoid organs, including brain [START_REF] Westermann | Naive, effector, and memory T lymphocytes efficiently scan dendritic cells in vivo: contact[END_REF]. In RABV-and CDV-infected mice, the presence of early-activated cells and T lymphocytes displaying high CRMP2 levels, associated with high migratory rates for T lymphocytes, suggests that the activation process may modulate T cell motility through CRMP2 modulation. Our recent observations support this hypothesis. An association between CRMP2 expression levels, T-cell activation status and in vitro cell migratory rates was observed in patients suffering from virus-induced neuroinflammatory disease [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF]. In addition, T-cell activation through CD3-CD28 stimulation clearly enhanced CRMP2 levels per cell (unpublished data). How CRMP2 operates in activated T-lymphocytes to drive their migration to the CNS is currently under investigation. The role of CRMP2 in the CNS provides some clues to understanding its function in the immune system. By its ability to associate with cytoskeletal elements, CRMP2 participates in the cytoskeletal reorganization required for neuronal cell reshaping and migration under semaphorin signals through the promotion of microtubule extensions [START_REF] Fukata | CRMP-2 binds to tubulin heterodimers to promote microtubule assembly[END_REF], a process inactivated by CRMP2 phosphorylation (Yoshimura et al., 2005;[START_REF] Cole | Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo[END_REF][START_REF] Uchida | Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease[END_REF]. In immune cells, we have shown the ability of CRMP2 to bind vimentin and tubulin [START_REF] Vincent | A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration[END_REF] thus to participate in lymphocyte reshaping. Inflammatory molecules, notably chemokines, could act as modulators of CRMP2 function, promoting T cell reshaping and motility in neuroinflammatory situations. This idea is supported by the role and distribution of ckemokines in the inflamed/infected brain. Indeed, chemokines are required for lymphocyte chemotaxis through actomyosin/ microtubule organization (Vicente-Manzanares and Sanchez-Madrid, 2004; [START_REF] Rey | Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes[END_REF]. Chemokines regulate cell entry into the brain microvasculature, direct navigation across the Virchow-Robin perivascular space and facilitate T cell crawling in brain parenchyma [START_REF] Kleine | Immune surveillance of the human central nervous system (CNS): different migration pathways of immune cells through the blood-brain barrier and bloodcerebrospinal fluid barrier in healthy persons[END_REF][START_REF] Lane | A central role for CD4(+) T cells and RANTES in virusinduced central nervous system inflammation and demyelination[END_REF][START_REF] Lee | Involvement of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1alpha in breast cancer cell migration through human brain microvascular endothelial cells[END_REF][START_REF] Liu | The T cell chemoattractant IFN-inducible protein 10 is essential in host defense against viralinduced neurologic disease[END_REF][START_REF] Ransohoff | Three or more routes for leukocyte migration into the central nervous system[END_REF]. Chemokines are expressed at high levels in the virus infected CNS, as it is the case after RABV infection [START_REF] Baloul | Apoptosis and rabies virus neuroinvasion[END_REF][START_REF] Roy | Failure to open the blood-brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection[END_REF][START_REF] Wang | Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system[END_REF].

In several infection models, the infiltration of activated T-lymphocytes into the brain is a wellcharacterized parameter linked to the development of encephalitis and neurodegeneration [START_REF] Irani | Isolation of brain parenchymal lymphocytes for flow cytometric analysis. Application to acute viral encephalitis[END_REF][START_REF] Khuth | Morbillivirus infection of the mouse central nervous system induces region-specific upregulation of MMPs and TIMPs correlated to inflammatory cytokine expression[END_REF][START_REF] Marten | Kinetics of virus-specific CD8+ -T-cell expansion and trafficking following central nervous system infection[END_REF]. A priming process initiated by a low number of infiltrating encephalitogenic T-lymphocytes could set the stage for a second greater wave of invading effector cells, as observed in multiple sclerosis patients and associated animal models [START_REF] Engelhardt | The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms[END_REF][START_REF] Norman | Mechanisms of lymphocyte migration in autoimmune disease[END_REF]. The subsequent induction of a shift towards a proinflammatory CNS microenvironment may have beneficial/unfavorable consequences, leading to viral clearance but promoting immune-related damage to neural cells [START_REF] Fujinami | Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease[END_REF][START_REF] Griffond | Specific alteration of the expression of selected hypothalamic neuropeptides during acute and late mouse brain infection using a morbillivirus: relevance to the late-onset obesity?[END_REF]. Whether or not T cells contribute to immunopathology in RABV is still controversial and seems to depend upon strain pathogenicity (Camelo et al., 2001b;[START_REF] Galelli | Abortive rabies virus central nervous infection is controlled by T lymphocyte local recruitment and induction of apoptosis[END_REF][START_REF] Hooper | The role of immune responses in the pathogenesis of rabies[END_REF][START_REF] Lafon | Modulation of the immune response in the nervous system by rabies virus[END_REF]. In CDV-infected mice, clinical and histological changes in the CNS do not develop immediately upon viral inoculation but only after a latency period, coinciding with T lymphocyte infiltration (Khuth et al., 2001). This suggests that infiltrating immune cells are important effectors of the intrathecal deleterious process translated in clinical manifestations.

The association we observed between CRMP2 expression in peripheral immune cells, in particular the presence of CRMP2hi cells, and the clinical scores displayed by encephalitic infected mice and the fact this is restricted to the infection of brain (not seen after the non neurovirulent RABV infection)

suggest that CRMP2 could be used as a cell marker to isolate and profile the cells with the potential to infiltrate the infected CNS. This could help in decrypting the association of migrating lymphocytes with the complex process leading to lethal encephalitis or recovery. Bearing early-activated T-lymphocyte and dendritic cell markers, CRMP2hi cells could comprise active cytolytic CD8+ T-lymphocytes, regulatory CD4+ T cells and antigen-presenting cells, each displaying their own role. They could therefore be crucial either in controlling the immune response to pathogens and virus clearance or in preventing reactivity to self-antigens, thus reducing the autoimmune reaction within the CNS [START_REF] Serafini | Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis[END_REF][START_REF] Serafini | Dendritic cells in multiple sclerosis lesions: maturation stage, myelin uptake, and interaction with proliferating T cells[END_REF][START_REF] Esiri | The interplay between inflammation and neurodegeneration in CNS disease[END_REF][START_REF] Lauterbach | Adoptive immunotherapy induces CNS dendritic cell recruitment and antigen presentation during clearance of a persistent viral infection[END_REF][START_REF] Ramakrishna | Vaccine-induced memory CD8+ T cells cannot prevent central nervous system virus reactivation[END_REF].

Detailed analysis of selected CRMP2hi cells along the course of viral infection and disease would

bring information on the nature and function of cells sequentially recruited to the inflamed CNS.

To conclude, the present work supports the idea that viral infection of the CNS enhances the ability of activated T-lymphocytes to switch on the intracellular machinery that recruits the cytoskeletal reorganizer CRMP2 and triggers cell migration. This also identifies CRMP2 molecule as a potential frequency in T cell zones of secondary lymphoid organs does not depend on LFA-1 expression and facilitates survival of effector T cells. J Immunol 174, 2517-2524.

Yoshimura, T., Kawano, Y., Arimura, N., Kawabata, S., Kikuchi, A., Kaibuchi, K., 2005. GSK-3beta reg

Legends

  CDV infected SV129 mice, PBMC and lymph nodes were sampled at different times(day 3, 7, 11 and 14) following injection of neuro-adapted virus (infected-mice) or brain suspension (sham-inoculated mice). At first, a significant decline was detected in the CD3+ lymphocyte frequency in PBMC on day 7 post virus-inoculation (47.2% versus 63.3% in sham mice), probably due to the immunosuppressive effect of CDV (Figure2-A). Nevertheless and similarly to what was observed in RABV infected mice, the frequency of CD69+ cells among PBMC increased (10.5% versus 5.2% in sham mice on day 7 post-inoculation). When CRMP2 expression was analyzed in terms of CRMP2+ cell frequency (% of positive cells in the total population), CDV infection did not modify the % of CRMP2 expressing cells (≈98% positive cells, not shown). In contrast, when the CRMP2 level per cell (mean fluorescence intensity= MIF) was analyzed in double stained cells (Figure2-B), CRMP2 expression showed an increase in MIF in CD3+ and CD69+ cells of CDV-infected mice, reaching significance on day 7 postinoculation. Focusing on the CD69+CRMP2+ subpopulation (Figure2-C), cytofluorimetry analysis indicated that CDV infection triggered the expansion of a discrete population characterized by a strong CRMP2 and CD69 expression. This cell subset, termed CRMP2hi, was recruited preferentially among the activated cell population CD69+, was characterized by an elevated CRMP2 expression (MIF for CRMP2hi cell population= 26-45 vs 16-20 for non-CRMP2hi cells) and constituted 57±13% of the CD69+CRMP2+ cell population and so, probably participated to the elevated mean fluorescence intensity detected in CD69+ activated cells of infected mice. CRMP2hi cells were specifically detected in PBMC and lymph node of the infected animals, at the peak of encephalitis, namely on day 7 (in 9 out 12 infected mice analyzed) and on day 11 post-inoculation (in 4 out 5 infected mice analyzed) (Figure2-D). Thus, even under conditions of CRMP2 constitutive expression, CDV infection increased CRMP2 level in a subset of activated cells in blood and lymph nodes.In conclusion, analysis of CRMP2 expression in peripheral immune cells performed in different models of viral infection indicated that neurotropic strains of CDV and RABV specifically increased the level of CRMP2 in circulating lymphocytes. Depending on the constitutive level of CRMP2 expression, neural infection triggered the expansion of CRMP2+ or CRMP2hi cell populations. These CRMP2+ cells recruited mainly among CD3+ T lymphocytes, notably in CD69+ early-activated cells.Selective recruitment of CRMP2+CD3 T cells in neurovirulent RABV and CDV infected CNSAssuming that elevated CRMP2 expression may favor T cell motility and transmigration to inflamed tissue, we analyzed whether CRMP2+ cells expansion in the periphery after RABV or CDV inoculation correlated with trafficking to the CNS as the viral infection progressed in this tissue. Mononuclear cells were isolated from the CNS using Percoll gradients and phenotyped by cytofluorimetry, at different times after viral inoculation. In the RABV infection model with the neurovirulent strain CVS we focused our analysis on CD3+ T cells, which selectively migrate into the CNS of infected mice (Figure3-A). In the absence of infection and also in the first 5 days of CVS infection, CD3+ T cells could only be detected in minimal amounts (10-15% of the Percoll-collected cells), whereas later in the infection, CD3+ T cells constituted 30% (day 8) and up to 60% (day 12) of the migratory cells. Double staining analysis (CRMP2 and CD3) showed a recruitment of CRMP2+CD3+ T cells in the CNS on day 8 (27% of the Percoll-collected cells). This percentage was unchanged on day 12 (Figure3-B). The combination of the CRMP2+ Percoll collected cell population was further investigated by double staining of CRMP2 with markers chosen to roughly identify B-lymphocytes (B220), T-lymphocytes (CD3), monocyte/macrophages and microglia (CD11b), NK and dentritic cells (CD11c), on days 0, 5, 8 and 12 post-infection. At each time point, the total % of CRMP2+ cells was taken as 100 and the frequencies of B220, CD11C, CD11b and CD3 were calculated (Figure3-C). Under non-infected conditions, CRMP2+ cells could be sorted into CD11b (80%) and B220 (20%) in the brain. CVS infection modified these ratio, inducing a drastic increase in CD3+CRMP2+ cells (38% and 62% are T lymphocytes on days 8 and 12, respectively), a decrease of B220+CRMP2+ cells and the appearance of a CD11c+CRMP2+ cell population accounting for a small percentage of the CRMP2+ population (peak 12% on day 8). CD11b antigen, a marker of both macrophages and microglia, could not differentiate the resident microglia trapped in the Percoll preparation from infiltrated immune phagocytes.Cytofluorimetry analysis of Percoll-collected cells was further performed in the mouse model of CDV infection on days 7 and 14 post-infection. Compared to non-infected conditions, the frequency of CD3+ T-lymphocytes and early-activated CD69+ cells detected in the brain infiltrate was significantly enhanced under CDV infection (reaching significance on day 14) (Figure4-A), indicating that in the course of CNS infection, inflamed brain attracts immune cells. Co-detection of CRMP2 with CD69 and CD3 antigens on migratory cells showed that almost all expressed high CRMP2 levels, thus behaving like CRMP2high cells (illustration of one infected-brain and sham-inoculated brains in Figure4-B).Immunocytochemistry performed on brain slices from mice examined on day 14 post-inoculation confirmed that CRMP2+ cells mainly infiltrated the infected neural tissue (Figure5-A) and showed the accumulation of CD4+ T lymphocytes in the parenchyma of several brain structures previously demonstrated to replicate CDV, including hippocampus and thalamus[START_REF] Bernard | Brain structures selectively targeted by canine distemper virus in a mouse model infection[END_REF] (Figure5-B). T lymphocytes, in particular, were present in brain parenchyma, at the proximity of structures involved in blood-CNS exchange: the velum interpositum of meninges, the choroid plexuses in ventricle. Immunofluorescence assay detected CD4+ and CD8+ cells in ethanol fixed neural tissue, confirming the presence of T lymphocytes in the brain infiltrate of CDV infected mice (Figure5-C). Codetection of CRMP2 and CD4 or CD8 antigens on brain slices could not be performed (different tissue fixation requirement). However, CRMP2 and TCR beta chain positive cells were co-detected in same brain areas (acetone fixation) (Figure5C) confirming the presence of CRMP2+ lymphocytes in infected/inflamed brain.

Figure 7 -

 7 Figure 7-A. To analyze the time course of CRMP2 expression in the peripheral immune cells, together with the clinical score, RABV infected mice were bled and sacrificed at different times post infection and individual clinical scores at the time of sacrifice were recorded. CRMP2 expression levels in PBMC and lymph node cells were then evaluated by cytofluorimetry. Similar to the CDV infection model, CRMP2hi cells were detected in infected mice, as shown by the high CRMP2 expression in the CD3+ T lymphocyte population of infected compared to non-infected mice (Figure 7-B). When CRMP2hi cells were plotted against the individual clinical score displayed by encephalitic mice (Figure 7-C), they appeared preferentially in sick mice displaying high clinical scores (>3-5). Indeed, a significant correlation between the cumulative clinical scores displayed by RABV-infected mice and the frequency of CRMP2+ cells in blood (R 2 = 0.69) was detected using a logarithmic representation of non-linear regression analysis (Figure 7-D). After infection with the neuronotropic RABV strain, PV,

Figure 1 :

 1 Figure 1: CRMP2 expression in peripheral blood mononuclear cells of RABV infected mice. A-After infection in the periphery with the neurovirulent RABV strain CVS (A,B,C) or with the neurovirulent and partially attenuated RABV strain PV or non neurovirulent RABV strain ERA (D), PBMC were collected from infected (black or grey histogram) and sham-infected (white histograms) mice 1, 7 and 9 days post infection (pi). Kinetics of CD3, CD69 and CRMP2 frequency among total viable PBMC (A and D), % of CD3 or CD69 positive cells among CRMP2 positive cells (B) and % of CRMP2+ cells among CD3 and CD69 positive cells (C) was followed by cytofluorimetry analysis using appropriate pairs of antibodies. A, B, C-infection with a neuronotropic CVS RABV strain causing fatal rabies. CRMP2+ cells recruit in CD3+ T lymphocytes and early activated immune cell populations.RABV infection triggers CRMP2 expression in CD3 T lymphocytes; virtually all CD69+ cells express CRMP2. D-Infection with a neuronotropic RABV strain, PV, causing abortive rabies (dark gray histograms, or infection with a non neuronotropic RABV strain ERA causing no CNS disease (light gray histograms). In contrast to the neuronotropic PV strain, ERA strain does not trigger the expansion of CRMP2+ in PBMC of infected mice. These data are representative of at least three independent experiments (±SD).

Figure 2 :

 2 Figure 2: CRMP2 expression in peripheral blood mononuclear cells of CDV infected mice. After infection of brain with CDV (gray histograms) or sham-infection (white histograms), PBMC were collected and analyzed by cytofluorimetry as decribed above. A-Transient reduction of CD3+ T cells and elevation of early-activated immune cells CD69+ frequency induced by brain infection; B-Elevated CRMP2 expression in CD3+ T cells and early-activated CD69+ cells, as shown by increased Mean Fluorescence Intensity (MFI); C-Detection in infected mice of early-activated CD69+ cells with elevated CRMP2 expression (MFI≥twice MFI observed in sham-infected mice) called CRMP2hi; D-Expansion of CRMP2hi cells increased progressively as infection and disease progressed. These analyses are representative of three independent experiments (±SD).

Figure 3 :

 3 Figure 3: Immune cells infiltration in the CNS of neurovirulent RABV CVS strain infected mice analyzed by cytofluorimetry. Cells infiltrating the CNS of CVS infected (gray histograms) and shaminfected (white histograms) were isolated by Percoll gradients, stained with appropriate pairs of antibodies and analyzed by cytofluorimetry. Kinetics of CD3+ T cell frequency (A) and double stained CD3 and CRMP2 cells (B) were followed 1, 5, 8 and 12 days after infection in infected (black histograms) and non-infected (white histograms) mice. C-The distribution of CRMP2 expressing cells among CD3, CD11c, CD11b and B220 positive cells from the CNS was analyzed in the course of RABV infection (5, 8 and 12 days pi) and compared to the non-infected control (day 0). T lymphocytes constitute the majority of infiltrated immune cells on day 12 post inoculation, while CD11c+ cells (corresponding roughly to dendritic cells) peaked on day 8. Each point of the curve includes two to three mice. Data were representative of three different experiments (±SD).

Figure 4 :

 4 Figure 4: Immune cell infiltration in the CNS of CDV infected mice analyzed by cytofluorimetry.Cells infiltrating the CNS were collected (7 and 14 dpi) and analyzed by cytofluorimetry as described above. A-Elevated recruitment of early activated CD69+ and CD3+ T cells under viral infection (black

Figure 5 :

 5 Figure 5: Analysis of the brain infiltrates in CDV-infected mice. Immunodetection of brain infiltrating immune cells was performed by immunofluorescence (A and C, brain removed andimmediately frozen) and by DAB staining (B, mice perfused with PFA) on coronal brain sections using anti-CD4, CD8, CRMP2 and TCR beta chain antibodies. A-CRMP2+ infiltrating cells are more abundant in the brain of CDV-infected than sham-inoculated mice (microscope field x400). B-CD4+ T lymphocytes infiltrated in CDV-infected brain structures hippocampus, thalamus[START_REF] Khuth | Morbillivirus infection of the mouse central nervous system induces region-specific upregulation of MMPs and TIMPs correlated to inflammatory cytokine expression[END_REF] and at proximity of the blood-CNS (velum interpositum) and CSF-CNS (third ventricle) interfaces (avidiin-biotin peroxidase system) (microscope field x100 and x400). C-Identification in CDV infected brain of CD4+ and CD8+ lymphocytes (microscope field x200) and CRMP2 expression by T lymphocytes (TCR beta chain positive cells) (microscope field x400).

Figure 6 :

 6 Figure 6: Association of elevated CRMP2 expression with high migratory rate of lymphocytes collected in CDV infected mice: CRMP2 expression was evaluated by cytofluorimetry in lymph node cells of CDV-infected compared to non-infected mice prior to migration tests were performed in Transwell system (B) and on hippocampus organotypic culture (C). A-Elevated CRMP2 expression level (MFI) in lymphocytes of CVD-infected mice. B-Elevated migration of lymph node cells of CDVinfected mice (3 non-infected versus 3 CDV-infected mice) in a Transwell system (3m pore membrane) in the presence of a chemokine gradient (CXCL12 -10ng/ml, CCL5 -100ng/ml, CCL2 and CXCL10 -20ng/ml, 37°C, 2h) (±SD). C-Migration analysis of lymph node cells stained with CFSE then spotted close to CDV-and non-infected hippocampus slices (18h contact then cell counting): elevated migratory rate of lymphocytes of CDV-infected versus sham-inoculated mice. Blockade experiment: treatment of lymphocytes with anti-CRMP2 antibody prior migration assay impeded tissue infiltration both on CDV-infected and non-infected hippocampus slices (open marks in graph ).

Figure 7 :

 7 Figure 7: Peripheral expansion of CRMP2hi cells in RABV infected mice correlates with clinical score: A-After infection, RABV infected animals develop clinical symptom which aggravate as the virus infectionprogresses into the NS. Progression was evaluated by scoring mobility and mortality as follows: 0=normal mice, 1=ruffled fur, 2=loss of agility, 3=one paralyzed hind leg, 4=two paralyzed hind legs, 5=total loss of mobility and 6=death. A representative evolution of the cumulative clinical scores (addition of individual scores of 8 animals per group); B-CD3+ T cells expressing strong CRMP2 fluorescence (CRMP2 high) can be observed in lymph node cells of infected animals (right panel showing CD3/CRMP2 expression in lymph nodes of 7 day RABV infected mice). These cells were absent in the CD3+ T cells of non-infected mice (middle panel) (representative data). C-CRMP2hi cells can be detected not only in lymph nodes but also in PBMC as the disease (individual clinical score on x axis) progresses (4 mice represented); D-When individual clinical scores were plotted against % of CRMP2+CD3+ T cells in lymph node, a significant correlation was observed (R= 0.69) between CRMP2 cells and individual clinical scores (logarithmic representation of non-linear regression analysis).

  Figure-1, Vuaillat et al

  in immune cells in healthy C57B6 and SV129 mice was different. While in healthy BALBC mice, less than 20 % of PBMC and lymph node mononuclear cells expressed CRMP2, virtually all PBMC and lymph node mononuclear cells of SV129 mice expressed CRMP2 (98.6±0.7% positive cells). To investigate how RABV and CDV infections regulate CRMP2 in these two strains of mice, CRMP2

expression was investigated by cytofluorimetry in PBMC and lymph nodes mononuclear cells of BALB/C and SV129 mice during the course of RABV or CVD infection, respectively.

marker of activated and motile T-lymphocytes generated during viral infection and potentially recruited in the inflamed CNS.