[Truncated abstract] Melanoma cell adhesion molecule (MCAM) is highly expressed in more than 70% of metastatic melanoma and is correlated with invasive potential. However, the specific contribution MCAM makes to invasion and metastasis in melanoma is not clear. In this study, I have demonstrated that transfection of MCAM into MCAM-negative melanoma and CHO cells leads to changes in cell shape, and the modulation of cell-to-cell and cell-matrix interactions. MCAM positive cells were slower to spread on collagen type I, collagen type IV and laminin 1 than MCAM negative cells, although these differences were not apparent on vitronectin, fibronectin and laminin 10. In contrast, MCAM expression had little effect on cell adhesion to any of the matrices tested. MCAM positive (compared to negative) cells also showed morphological changes and a rearrangement of the actin cytoskeleton when plated on a matrix containing laminin 5. Taken together, these data suggest that MCAM expression modulates β1-integrinmediated spreading on matrix, but has little effect on αvβ3-mediated cell-matrix interactions. As this study provided little evidence to suggest that MCAM transfection altered β1 integrin expression levels on melanoma cells, it is proposed that a competitive interaction between the cytoplasmic domains of MCAM and β1 integrin may affect mature focal adhesion assembly. MCAM expression in melanoma cells was also associated with decreased cell movement over matrix into a scratch-wound site and an increased tendency to form cell cords on Matrigel. These two assays gauge the propensity of a cell to engage in cell-cell versus cell-matrix interactions, and suggest that MCAM positive cells favour cell-cell adhesion. Interestingly, MCAM transfection was also associated with an increased ability of melanoma cells to migrate through a basement membrane towards a chemoattractant. ... Analysis of the intracellular domain of MCAM revealed the presence of tyrosine and dileucine endocytosis signals. Interestingly, disruption of these two motifs did not seem to impair the internalization of MCAM from the cell surface. The di-leucine motif, however, was necessary for the recycling of MCAM back to the surface following endocytosis. Lastly, MCAM was found to exists as dimers within the cell membrane in the absence of ligand, although the exact location of the dimerization motif is not yet clearly defined. Collectively, findings from my study suggest: MCAM expression in melanoma cells facilitates cell-cell interactions, whilst concomitantly modulating cell-matrix interactions. MCAM transfection also leads to enhanced migration of melanoma cells through a basement membrane. Thus, MCAM expression may increase the ability of melanoma cells to migrate as a collective, a feature of highly invasive cancer. The intracellular domain of MCAM interacts with ApxL2, a novel member of the Shroom family of actin-binding proteins. It is likely that ApxL2 links a proportion of MCAM within the cell to the actin cytoskeleton, contributing to cell shape determination and other processes, such as migration. MCAM exists as dimers on the cell surface and is internalized at least partially by a clathrin-mediated mechanism.

Objectifs: Chez les patients atteints de sclérose en plaques (SEP), des lymphocytes pro-inflammatoires utilisent des molécules d’adhérence afin de parvenir à traverser la barrière hémo-encéphalique (BHE) et former des lésions multifocales dans le système nerveux central (SNC). Dans le contexte de la SEP, les lymphocytes CD4 auto-agressifs polarisés en TH17 (sécrétant de l’IL-17) sont reconnus comme contribuant à la formation des lésions. Le rôle des lymphocytes CD8 TC17 est quant à lui encore mal défini. L’identification de marqueurs de surface spécifiquement exprimés par les lymphocytes TH17 et TC17 faciliterait la caractérisation de ces sous-populations pathogéniques et fournirait de nouvelles cibles thérapeutiques pour traiter la SEP. Méthodologie: Nous avons identifié MCAM lors d’analyses protéomiques de cellules endothéliales de la BHE humaine et de lymphocytes T humains. Nous avons caractérisé le phénotype et la fonction de ces cellules exprimant MCAM ex vivo, in vitro, in situ et in vivo, à partir de matériel obtenu de témoins (contrôles), de patients atteints de SEP et d’animaux atteints d’encéphalomyélite auto-immune expérimentale (EAE). Résultats: MCAM est exprimé à la fois par les cellules endothéliales de la BHE humaine et par une sous-population de lymphocytes T effecteurs mémoire CD161+ et CCR6+. Les lymphocytes CD4 et CD8 MCAM+ expriment plus d’IL-17, IL-22, GM-CSF et granzyme B (Gz B) que les lymphocytes MCAMneg. De plus, l’expression de MCAM est fortement augmentée à la surface des lymphocytes T CD4+ et CD8+ lors des poussées de SEP, alors que les traitements immunomodulateurs en diminuent l’expression. In situ, l’expression de MCAM par les cellules endothéliales de la BHE est plus marquée au site des lésions de SEP et d’EAE, et on retrouve des lymphocytes CD4 et CD8 MCAM+ au sein de ces infiltrats périvasculaires du SNC. In vitro, les lymphocytes CD8 MCAM+ causent plus de mort oligodendrocytaire et bloquer MCAM diminue la transmigration des CD8 TC17 et des CD4 TH17 à travers les cellules endothéliales de la BHE humaine. In vivo, dépléter les lymphocytes CD4 ou CD8 MCAM+ améliore les signes cliniques de l’EAE par transfert. Par ailleurs, l’expression de MCAM est régulée à la hausse à la surface des lymphocytes CD4 et CD8 de la souris transgénique TCR1640, un modèle animal d’EAE spontanée. Finalement, bloquer MCAM atténue les déficits neurologiques chroniques aussi bien du modèle d’EAE induite avec le MOG35-55 que du modèle d’EAE spontanée. Conclusion: Nos données démontrent que les lymphocytes encéphalitogéniques produisant de l’IL-17 et présentant une capacité effectrice et migratoire marquée expriment MCAM. MCAM pourrait servir de biomarqueur en SEP et constituer une cible thérapeutique valable pour traiter les conditions neuroinflammatoires. / Objective: In multiple sclerosis (MS), pro-inflammatory lymphocytes use adhesion molecules to cross the blood-brain barrier (BBB) and accumulate in central nervous system (CNS) lesions. CD4 T lymphocytes polarized into auto-aggressive encephalitogenic TH17 (IL-17 secreting) are known to partake in MS lesion formation. Much less is known about the role of CD8 TC17. Identification of specific surface markers and adhesion molecules expressed by TH17 and TC17 lymphocytes would allow further characterization of these pathogenic subsets and would provide new therapeutic targets in MS. Methodology: We identified MCAM in a proteomic screen of human BBB endothelial cells (ECs) and on a subset of T lymphocytes. We characterized the phenotype and function of MCAM-expressing cells ex vivo, in vitro and in situ using human and mouse material obtained from controls, MS subjects and Experimental Autoimmune Encephalomyelitis (EAE) animals. Results: MCAM is expressed by human BBB-ECs and by human effector memory CD161+ and CCR6+ T lymphocytes. Both CD4 and CD8 MCAM+ lymphocytes express more IL-17, IL-22, GM-CSF and Gz B than MCAMneg lymphocytes. Moreover, MCAM is strikingly up-regulated in human on CD4+ and CD8+ T lymphocytes during MS relapses, while treatment decreases MCAM expression. In situ, MCAM+ CD8 and CD4 T lymphocytes are present in perivascular infiltrates of MS and EAE CNS specimens, while MCAM expression is up-regulated on BBB-ECs within lesions. In vitro, MCAM+ CD8 T lymphocytes display higher killing capacity of oligodendrocytes, and MCAM blockade reduces CD8 TC17 and CD4 TH17 transmigration across human BBB-ECs. In vivo, depletion of MCAM+ cells from reactivated CD4 T lymphocytes and from CD8 T lymphocytes decreases clinical symptoms in adoptive transfer EAE. Furthermore, expression of MCAM is up-regulated on CD4 and CD8 T lymphocytes in the TCR1640 transgenic mice, a model of spontaneous EAE. Finally, blocking MCAM in both MOG35-55-induced and spontaneous primary progressive EAE attenuates chronic neurological deficits. Conclusions: Our data demonstrate that encephalitogenic IL-17-producing lymphocytes with high effector and migratory capacity express MCAM, and that MCAM could serve as a biomarker for MS and a valuable target for the treatment neuroinflammatory conditions.