Дисертації з теми "Integrin LFA-1"

This work was undertaken to characterise the interaction of the leukocyterestricted integrin LFA-1 (CD1la/CD18) with its ligands. LFA-1 function is critical for an immune response and, for example, allows leukocyte binding and transmigration across the endothelium, antigen presentation and cytotoxic T lymphocyte-mediated killing. The ligands for LFA-1 are the Intercellular Adhesion Molecules (ICAMs), with ICAM-1, ICAM-2 and ICAM-3 being the best characterised. The binding sites on ICAM-1 and ICAM-3 for LFA-1 were investigated with the use of antibodies and mutated proteins. The following regions were found to have a role in binding LFA-1: the CFG face of ICAM-3 domain 1; domain 2 of ICAM-1; a residue in domain 1 of ICAM-1 that is mutated at high frequency in African populations and is associated with susceptibility to cerebral malaria. Binding of Mg2+ or Mn 2+ to the extracellular region of LFA-1 and intracellular signalling can both stimulate LFA-1 to adhere to ICAM-1, but by different processes. The former mechanism induces a high affinity form of LFA-1, which was shown to be achieved by an inter-domain movement involving the I domain of the LFA-1 a subunit. This is the first physical evidence for a conformational change occurring in an integrin upon activation. The mechanism by which intracellular signalling activates LFA-1 was demonstrated to involve calpaindependent clustering of LFA-1 in the membrane, thus increasing the avidity of LFA-1 for ICAM-1. Leukocyte Adhesion Deficiency-1 and Glanzmann's Thrombasthenia are genetic disorders in which mutations in the integrin genes result in absence of expression or expression of a non-functional integrin. The defects in function of leukocytes from a patient with clinical features of both disorders were studied. The results suggest that the patient has a novel form of integrin dysfunction in which integrins are expressed at normal levels, can be induced to bind their ligands by mechanisms which increase the affinity of interaction, but cannot be stimulated to bind ligand by intracellular signalling pathways.

Cellular adhesion plays a crucial role in the biological function of cells, allowing them to communicate and signal, as well as physically anchor, by enabling them to adhere to either other cells or the extra cellular matrix (ECM). This process is regulated by several factors including intrinsic bond kinetics, internal cellular signaling, environment, force exerted on the bond, and force history of the bond. Concerning the force and force history dependence, the observation of catch bonds in integrin binding has asked as more questions than it has answered. To explore the force and force history dependence this process, each bond was loaded to a peak force before relaxing to a much lower force that was held for the duration of the measurement. Two different integrins were studied, both of which have in previous works exhibited a catch bond. Furthermore, the effects of different metal ion conditions and an allosteric antagonist were also studied to elucidate the conformational effects on force priming of integrin. What was observed was that I domain, or αA domain, possessing integrin, whether tested against its more active or less active binding state, changed very little in terms of off rate once the priming force was applied. However in the I domain, or αA domain, lacking integrin, the observed off rate changed as well. It seems that force priming is capable of causing integrin to bind in a stronger manner regardless of the other conditions used to either activate or inhibit binding. However the way in which the binding is strengthened depends on the receptors structure.

Neuroinflammation is a complex inflammatory process occurring within the central nervous system (CNS). It is mediated by the production of cytokines, chemokines, reactive oxygen species and secondary messengers produced and secreted by CNS-resident cells and recruited immune cells. Recent studies highlighted the key role of neutrophils as crucial players in chronic inflammatory diseases including CNS disorders such as Alzheimer’s disease (AD) and multiple sclerosis (MS). Previous studies on experimental autoimmune encephalomyelitis (EAE), the animal model of MS, have shown that neutrophils in the CNS are localized in close proximity to damaged tissues, such as demyelinated areas and leaked vessels, suggesting their potential detrimental role in the pathogenesis and progression of MS/EAE. The extravasation of neutrophils into the CNS from the peripheral circulation is suggested to be induced by CNS-resident sentinel cells (e.g., astrocytes, resident macrophages and microglia), which are activated by tissue danger signals release. Some studies suggested that both astrocytes and microglia might regulate neutrophil functions. Inversely, CNS-invading neutrophils could release factors that affect reactivity of glial cells. The aim of this project is to shed light on the cellular interplay exerted by infiltrating neutrophils in the spinal cord (SC) during EAE, taking advantage in particular of our cutting-edge live imaging techniques. At first, flow cytometry studies carried on EAE mice showed neutrophil infiltration during all the clinical phases of disease but especially at the peak, during which a massive neutrophil infiltration within the parenchyma was observed. Immunofluorescence staining on SC sections revealed also numerous extravasated neutrophils establishing physical contacts with astrocytes and microglia/macrophages, suggesting that neutrophil-glial cell crosstalk may represent a previously unknown key axis during EAE. To corroborate this hypothesis, we developed a protocol of target validation on a dynamic in vitro neutrophil-glial cell co-culture system. We observed that neutrophils display different motility parameters when co-cultured with astrocytes or microglia/macrophages, and that Leukocyte function associated antigen-1 (LFA-1) blockade affected exclusively interactions with myeloid cells when isolated at the peak of disease. These data were also confirmed using neutrophils isolated from LFA-1 knock-out mice. Interestingly, we obtained similar results in vivo performing two-photon laser microscopy in the subarachnoid space (SAS) of lumbar SC at the peak of disease. Weakly motile extravasated neutrophils were found, moving preferentially located near to the vessels. Interestingly, the blockade of LFA-1 led to a progressive reduction in the portion of neutrophils that were moving slowly near the blood vessels, resulting in an increased portion of cells that, once left vessel compartmentalization, drastically enhanced velocity and directness moving deep in spinal SAS space. In CX3CR1-eGFP EAE mice, the local administration of anti-LFA-1 antibody clearly inhibited perivascular macrophage-neutrophil interactions, suggesting that LFA-1 integrin is a key integrin controlling neutrophil dynamics at the interface with SC parenchyma during acute EAE. To check a therapeutic relevance of our in vitro and in vivo findings, we performed intrathecal injection of anti-LFA-1 antibody at disease onset and 4 days later in EAE-induced mice. We observed that anti-LFA-1 treatment determined a significant inhibition of EAE clinical symptoms, together with amelioration in neuropathological hallmarks, in terms of infiltrated leukocytes, demyelination, microgliosis and astrogliosis. Collectively, the molecular mechanisms controlling neutrophil crosstalk with glial cells could represent an innovative field of study, thus contributing to develop new therapeutic strategies for the treatment of MS.

L’Alzheimer è una severa e progressiva malattia neurodegenerativa età-dipendente, dalla eziologia sconosciuta. Colpisce 35 milioni di persone nel mondo e porta ad un deterioramento cognitivo, dovuto alla perdita neuronale in quelle regioni del cervello deputate all’apprendimento e alla memoria. Le due principali e distintive caratteristiche neuropatologiche della malattia di Alzheimer sono i depositi extracellulari di peptide beta-amiloide (placche) e i filamentosi aggregati intracellulari di proteina tau iperfosforilata. Recenti studi suggeriscono che la neuroinfiammazione può rappresentare un segno distintivo della malattia e una potenziale forza trainante nell’ Alzheimer. I meccanismi infiammatori coinvolti nella patogenesi rimangono largamente sconosciuti e una migliore conoscenza del ruolo dell’infiammazione nella malattia di Alzheimer può essere d’aiuto nello sviluppare nuovi approcci terapeutici per rallentare la progressione di questa malattia. Il presente studio ha esaminato il ruolo dell’infiammazione vascolare e del traffico leucocitario, in particolare il ruolo dei neutrofili, in modelli murini della malattia di Alzheimer e come queste cellule sono connesse a deficits comportamentali e alla neuropatologia. Studi di microscopia confocale hanno mostrato un progressivo incremento nell’espressione di ICAM-1, VCAM-1, E- e P-selectin, e un elevato numero di cellule infiammatorie nel cervello di topi 5XFAD e 3xTg-AD, modelli murini di Alzheimer, durante le fasi iniziali della malattia, quando gli animali inziano a prensentare deficits cognitivi nei tests comportamentali. Sorprendentemente, abbiamo trovato che una larga parte di leucociti infiltranti erano neutrofili, suggerendo che queste cellule possono giocare un ruolo nella patogenesi della malattia. Inoltre, studi di microscopia confocale ha rilevato che i neutrofili producono NETs, trappole neutrofiliche extracellulari, suggerendo che queste formazioni possono rappresentare un nuovo meccanismo di malattia nell’Alzheimer. Esperimenti di microscopia a due fotoni ha confermato l’abilità dei neutrofili di invadere efficientemente la corteccia cerebrale e diffondere in gran numero nel parenchima, divenendo potenzialmente dannosi per le cellule neurali. Con interesse, abbiamo osservato che oligomeri solubili di beta-amiloide 1-42 erano in grado di attivare una rapida adesione integrino-dipendente su fibrinogeno umano e ICAM-1 e l’affinità dell’integrina LFA-1 nei neutrofili. In particolare, la deplezione neutrofilica così come il blocco del traffico dei neutrofili mediante anticorpo anti-LFA-1 durante le fasi iniziali della malattia, ha portato all’inibizione dei deficits cognitivi nei topi 5XFAD e 3xTg, suggerendo che i neutrofili hanno un ruolo chiave nel danno cerebrale e nei deficits cognitivi nella malattia di Alzheimer. Inoltre, abbiamo trovato che l’inibizione della funzione del neutrofilo fortemente riduce l’attivazione microgliale e il deposito di beta amiloide, suggerendo che i neutrofili giocano un ruolo chiave nella progressione della malattia. Noi abbiamo anche valutato campioni corticali cerebrali umani da pazienti con la malatttia di Alzheimer e abbiamo osservato un elevato numero di neutrofili aderenti e che diffondevano nelle venule cerebrali, che migravano nel parenchima cerebrale e producevano NETs, paragonandoli a soggetti controllo, indicando così un possibile ruolo per i neutrofili anche nella malattia umana. In conclusione, i nostri risultati suggeriscono che alle fasi iniziali della patogenesi della malattia di Alzheimer c’è un attivo processo infiammatorio, che include l’aumento dell’espressione di molecole di adesione sull’endotelio cerebrovascolare e la migrazione transendoteliale dei leucociti nel cervello, in particolare il reclutamento dei neutrofili. Ciò, ulteriormente, promuove danni vascolari e disfunzioni di cellule neurali, contribuendo a deficits cognitivi e alla progressione della malattia di Alzheimer. Quindi, i neutrofili possono avere un ruolo cruciale nella patologia e l’inibizione del traffico neutrofilico può rappresentare un nuovo e potenziale target terapeutico nella malattia di Alzheimer.
Alzheimer’s disease (AD) is an age-related, severe and progressive neurodegenerative disorder with unknown etiology. It affects 35 million people worldwide and leads to cognitive deterioration, due to neuronal loss in brain regions involved in learning and memory. The two main neuropathological hallmarks of AD are: extracellular deposits (plaques) of amyloid-beta peptide (Aβ) and filamentous intracellular aggregates (neurofibrillary tangles) of hyperphophorylated tau protein. Recent data suggest that neuroinflammation may represent a new hallmark of disease and a potential driving force in AD. However, the inflammation mechanisms involved in AD pathogenesis remain largely unknown and a better understanding of the role of inflammation in AD may help to develop new therapeutic approaches to slow the progression of this disorder. The present study examined the role of vascular inflammation and leukocytes trafficking, in particular the role of neutrophils, in murine AD models and how these cells related to behavioral impairments and neuropathology. Confocal microscopy studies showed a progressive increase in the expression of ICAM-1, VCAM-1, E- and P-selectin and high numbers of inflammatory cells in the brain of in 5XFAD and 3xTg-AD mouse models of AD during early phase of disease when animals start to present cognitive deficit in behavioral tests. Surprisingly, we found that a large part of the infiltrating leukocytes were neutrophils, suggesting that these cells may play a role in disease pathogenesis. Moreover, confocal microscopy studies revealed that neutrophils produce neutrophil extracellular traps (NETs), suggesting that NET formation may represent a new disease mechanism in AD. Two-photon microscopy experiments confirmed the ability of neutrophils to efficiently invade the cortex and diffuse in high numbers in the parenchyma being potentially harmful to neural cells. Interestingly, soluble amyloid-beta 1-42 oligomers were able to trigger rapid integrin-dependent adhesion to human fibrinogen and ICAM-1 and LFA-1 affinity in neutrophils. Notably, neutrophil depletion as well as the blockade of neutrophil trafficking by anti-LFA-1 integrin antibody during early phase of disease, led to the inhibition of cognitive deficits in 5XFAD and 3xTg-AD mouse models, suggesting that neutrophils have a key role in brain damage and cognitive deficit in AD-like disease. Moreover, we found that inhibition of neutrophil function strongly reduced microglial activation and amyloid deposition, suggesting that neutrophils play a key role in disease progression. We also evaluated human cortical brain samples from subjects with AD and observed elevated numbers of neutrophils adhered and spread inside brain venules and migrated in the parenchyma and produced NETs compared to control subjects, indicating a possible role for these cells in humans as well. In conclusion, our results suggest that, starting at the early stage of AD, there is an active inflammatory process, which includes up-regulation of adhesion molecules on cerebrovascular endothelium and leukocyte trans-endothelial migration into the brain, particularly neutrophil recruitment. This further promotes vascular damage and neural cell dysfunction, contributing to cognitive deficits and AD progression. Therefore, neutrophils may play a pivotal role in the AD pathology and inhibition of neutrophil trafficking may represent a novel potential therapeutic target in AD.

La rapida induzione dell’affinità integrinica è un processo dinamico cruciale nel reclutamento leucocitario, che è controllato da complessi meccanismi molecolari di segnale intracellulare indotti da chemochine. Le small GTPasi della famiglia di rho e rap sono certamente le molecole di segnale più studiate in questo contesto; dati recenti da noi ottenuti hanno evidenziato inoltre un ruolo importante delle proteine tirosin-chinasi della famiglia delle Jak (Jak PTKs) che agiscono da regolatori a monte delle small GTPasi. Gli scambiatori di nucleotidi guanosinici (GEFs-Guanosine Exchange Factors) sono i principali attivatori diretti delle small GTPasi e quindi rappresentano le molecole canditate più probabili per chiarire il legame funzionale tra Jak PTKs e il modulo della rho. In questo studio abbiamo dimostrato il ruolo regolatorio concorrente di quattro differenti rho-GEFs Vav1, Sos1, Arhgef1 e Dock2 nella modulazione dell’affinità dell’integrina LFA-1 e nella conseguente adesione cellulare di linfociti T umani primari stimolati con la chemochina CXCL12. La ridotta espressione di queste quattro molecole porta ad una minore induzione dell’affinità dell’integrina LFA-1 e ad una ridotta adesione all’ICAM-1 in condizioni statiche e sotto flusso. Da notare, l’attivazione di queste quattro proteine, indotta da CXCL12, è mediata dalle Jak PTKs e avviene in un intervallo di tempo coerente con la rapida induzione dell’affinità integrinica da chemochine. Inoltre l’attivazione di RhoA e Rac1 è strettamente dipendente dall’attività di Vav1, Sos1, Arhgef1 e Dock2. Complessivamente in questo studio abbiamo identificato e caratterizzato dettagliatamente il ruolo regolatorio di quattro rho-GEFs nell’adesione mediata da LFA-1 indotta da CXCL12, fornendo una descrizione completa dei meccanismi molecolari di segnale esistenti tra Jaks e modulo della rho. Analizzando i nostri dati da un punto di vista quantitativo, abbiamo riscontrato alcune differenze tra queste proteine osservando un ruolo più marcato per Vav1 e Sos1 in confronto a quello di Arhgef1 e Dock2. Questo diverso coinvolgimento di molteplici rho-GEFs con apparentemente la stessa funzione può avvalorare la nuova interpretazione quantitativa dei meccanismi di trasduzione del segnale, dove la complessità a livello molecolare è essenziale per generare un sistema flessibile in grado di rispondere efficientemente a differenti condizioni ambientali.
The rapid integrin affinity up-regulation is a crucial dynamic process in leukocyte recruitment that is controlled by a complex inside-out signalling pathway induced by chemokines. Small GTP binding proteins of rap and rho family are certainly the most studied signaling molecules involve in this pathway; in addition our recent data identified Jak PTKs as new upstream regulator of these small GTPases. Considering that Guanosine Exchange Factors (GEFs) are the main direct activators of small GTPases, they represent obvious molecule candidates to fill out the functional gap between Jak PTKs and rho-module. In this study we show the concurrent regulatory role of four rho specific GEFs Vav1, Sos1, Arhgef1 and Dock2 in CXCL12-induced LFA-1 affinity triggering and mediated-adhesion in human T lymphocytes. A reduced expression of these four molecules resulted in an impaired chemokine-induced LFA-1 affinity up-regulation and in a reduced cell adhesion to ICAM-1 in static and under-flow conditions. Importantly, CXCL12-activation of these four proteins is mediated by Jak PTKs and occurs in a time frame coherent with LFA-1 affinity triggering by chemokine. Moreover the activation of RhoA and Rac1 is strictly dependent on Vav1, Sos1, Arhgef1 and Dock2 activity. Collectively in this study we identified and fully characterized the role of four rho-GEFs in CXCL12-induced LFA-1 mediated adhesion providing a comprehensive signalling link between Jak PTKs and rho-module. Considering our results from a quantitative point of view, we observed some variability in the relative regulatory role of these proteins, with a major role for Vav1 and Sos1 with respect to Arhgef1 and Dock2 activity. This variable involvement of multiple rho-GEFs with apparently the same function may support the new emergent quantitative-concurrency view of signal transduction in which this complexity in mechanisms controlling integrin activation is essential to generate a very flexible signalling system able to efficiently respond to a variety of environmental conditions.

La Sclerosi Multipla (SM) è una patologia infiammatoria autoimmune cronica, disabilitante e demielinizzante, del sistema nervoso centrale (SNC). La migrazione di cellule T autoreattive e la loro riattivazione attraverso la presentazione antigenica effettuata dalle locali cellule presentanti l’antigene, rappresentano due eventi critici nella patogenesi della SM e nel suo modello animale, l’encefalite autoimmune sperimentale (EAE). Le risposte ai potenziali antigeni all’interno del SNC richiedono una migrazione a lungo raggio, comunicazione a corto raggio e contatti cellula-cellula diretti con le cellule presentanti l’antigene. Il principale obiettivo di questo studio è indagare il ruolo delle integrine L2 (LFA-1) e 4 (VLA-4 and 47) nella migrazione e nella motilità delle cellule autoreattive Th1 e Th17 in corso di EAE all’interno del midollo spinale utilizzando le moderne tecniche di microscopia intravitale. La microscopia intravitale permette di visualizzare questi processi dentro il midollo spinale, il quale rappresenta il principale sito di infiammazione durante l’EAE. Mediante la microscopia intravitale in epifluorescenza abbiamo prima di tutto investigato il ruolo delle integrine 4 e LFA-1 nella migrazione delle cellule autoreattive Th1 e Th17 all’interno dei vasi piali del midollo spinale durante la fase preclinica, l’esordio e la fase cronica di EAE. Abbiamo utilizzato un modello di EAE immunizzando topi C57BL/6 con il peptide MOG35-55. Le cellule Th1 e Th17 MOG-specifiche sono state prodotte in vitro da topi TCR-transgenici 2D2, marcate con label fluorescenti e iniettate in endovena nei topi immunizzati direttamente prima dell’acquisizione. I nostri risultati pongono l’accento su un ruolo selettivo per l’integrina LFA-1 nella migrazione delle cellule Th1 in particolare durante le prime fasi di malattia. Ruolo che invece non è svolto in fase cronica di malattia. Inoltre, il blocco della subunità 4, ma non dell’integrina 47 inibisce fortemente sia il rotolamento sia l’adesione stabile delle cellule Th1 all’endotelio infiammato. Questo suggerisce l’integrina VLA-4 come principale mediatrice della migrazione delle Th1 nel SNC infiammato in corso di EAE. Di notevole interesse è inoltre il selettivo ruolo per l’integrina 47 evidenziato in particolare nell’adesione stabile esclusivamente delle cellule Th17 all’esordio ed in fase cronica di malattia. Successivamente, per studiare la motilità intraparenchimale delle cellule Th1 e Th17 nel midollo spinale durante la fase preclinica della patologia, il picco di malattia e la fase cronica abbiamo utilizzato la microsocopia laser a due fotoni. I nostri risultati dimostrano una massiva infiltrazione di Th1 e Th17 nel parenchima del SNC al picco di malattia, mentre la migrazione di queste cellule durante le altre fasi della malattia è significativamente minore. In seguito, il nostro studio si è focalizzato sulla fase clinica della malattia e abbiamo osservato che le cellule Th1 e le cellule Th17 mostrano differenze significative nelle componenti direzionali, con le cellule Th1 che si muovono velocemente in direzione rettilinea, coprendo lunghe distanze nel parenchima del midollo spinale, mente le cellule Th17 girano intorno in un volume specifico del tessuto facendo stop and go. In particolare, il blocco dell’integrina LFA-1 influenza drasticamente le dinamiche delle cellule, portando ad una riduzione nella velocità e interferendo con il loro pattern di motilità rettilinea. Inoltre, in presenza di un anticorpo bloccante anti-LFA-1, le cellule Th17 mostrano una riduzione di velocità drastica. Diversamente, l’anticorpo anti-4 non ha nessun effetto sul comportamento motile delle Th1, ma riduce fortemente la velocità delle Th17 suggerendo che l’integrina VLA-4 non sia richiesta per la motilità intraparenchimale durante l’EAE. Tuttavia, alla luce dei risultati ottenuti in precedenza mediante microscopia intravitale, va preso in considerazione un selettivo ruolo esercitato dall’integrina 47 nella motilità intraparenchimale delle Th17. Complessivamente, i nostri risultati suggeriscono che l’LFA-1 sia la principale integrina che controlla la motilità delle cellule Th1 e Th17 e che 47 sia invece selettivamente coinvolta nella motilità delle cellule Th17 nel midollo spinale in condizioni infiammatorie al picco di malattia. A sostegno di questi risultati abbiamo testato un trattamento terapeutico mediante blocco locale, a livello intratecale, delle integrine LFA-1 e 47 nel nostro modello murino di EAE cronica. Entrambi i trattamenti hanno evidenziato una riduzione nello sviluppo della malattia che suggerisce l’importanza di interferire direttamente con le dinamiche delle cellule pro infiammatorie a livello del SNC. Approfondire dunque la conoscenza dei meccanismi molecolari che controllano la motilità intratissutale di cellule T attivate nel SNC potrebbe aiutarci a identificare nuove strategie terapeutiche per le patologie autoimmuni cerebrali croniche.
Multiple sclerosis (MS) is a chronic disabling autoimmune inflammatory demyelinating disease of the central nervous system (CNS). The migration of autoreactive T cells from the blood into the CNS and their reactivation through antigen presentation by local antigen presenting cells (APCs) represent critical events in the pathogenesis of MS and its animal model, the experimental autoimmune encephalomyelitis (EAE). The responses to potential antigens inside the CNS require long-range migration of cells, short-range communication and direct cell-cell contact with APCs. The main goal of this study was to investigate the role of L2 (LFA-1) and 4 (VLA-4 and 47) integrins in the migration and motility behavior of Th1 and Th17 cells, which represent key players in the induction of EAE, using intravital microscopy approaches. Intravital microscopy techniques allow the visualization of T cell migration and reactivation in the spinal cord (SC), which represents the main inflammation site during EAE. By using epifluorescence intravital microscopy (IVM) we first studied the roles of 4 and LFA-1 integrins in Th1 and Th17 cell adhesion in the pial vessels of spinal cord (SC) venules in mice immunized with MOG35-55 peptide during the preclinical phase, disease onset and chronic phase of disease. We used an EAE model by immunization of C57BL/6 mice with MOG35-55 peptide. MOG35-55-specific Th1 and Th17 cells were produced in vitro from 2D2 TCR transgenic mice, labeled with fluorescent dyes and intravenously injected in immunized mice before imaging. Our results underlined a selective role for LFA-1 integrin in Th1 cell recruitment in inflamed SC vessels during the early phases of the disease but not during the chronic phase. Moreover, blocking antibodies against the 4subunit, but not blockade of 47 integrin greatly inhibited rolling and firm adhesion of Th1 cells in the SC venules during all disease phases, suggesting that VLA-4 is the major molecule involved in Th1 cell adhesion in the SC venules during EAE. Interestingly, blockade of 47 integrin led to a significant reduction of firm adhesion in Th17 cells at the onset and chronic phase of EAE indicating a selective role of 47 integrin in the recruitment of Th17 cells in the inflamed CNS. Taking advantage of two-photon laser microscopy (TPLM) approach we next investigated the motility behavior of fluorescently labeled Th1 and Th17 cells within SC parenchyma during different disease phases. Our results showed a massive infiltration of Th1 and Th17 cells in the CNS parenchyma at disease peak, whereas the migration of these cells during other phases of disease was significantly lower. Furthermore, Th1 and Th17 cells displayed significant differences in the directional component, with Th1 cells moving faster in straight directions covering long distances deep in the SC parenchyma, whereas Th17 cells moved around in a specific volume of tissue in a stop and go mode. Notably, the blockade of LFA-1 integrin drastically affected the dynamics of Th1 cells leading to a reduction in velocity and interfering with their straight-line motility pattern. Moreover, Th17 cells displayed a drastic reduction of velocity in the presence of a blocking anti-LFA-1 antibody. The analysis of cellular morphology suggested that LFA-1 is actively involved in the cytoskeleton rearrangements necessary for T cell amoeboid migration inside the CNS, but had no role in the cytoskeleton dynamics in Th17 cells. Notably, 4integrins had no role in Th1 cells motility, but drastically reduced the dynamics of Th17 cells inside the SC parenchyma. To check the therapeutic relevance of our intravital microscopy findings, we performed intrathecal injection of anti-LFA-1 or anti-47 antibodies at disease onset and observed a significant inhibition of EAE progression in mice immunized with MOG35-55 peptide. Collectively, our data demonstrate that LFA-1 integrin differently controls intraparenchymal Th1 and Th17 cells dynamics, whereas 47 integrin is selectively involved in Th17 cell trafficking in the CNS during EAE. Furthermore, our results suggest that interfering with the molecular mechanisms controlling intraparenchymal dynamics of activated T cells may represent a new therapeutic strategy for CNS autoimmune diseases.