Multiple sclerosis endophenotypes identified by high-dimensional blood signatures are associated with distinct disease trajectories.

One of the biggest challenges in managing multiple sclerosis is the heterogeneity of clinical manifestations and progression trajectories. It still remains to be elucidated whether this heterogeneity is reflected by discrete immune signatures in the blood as a surrogate of disease pathophysiology. Accordingly, individualized treatment selection based on immunobiological principles is still not feasible. Using two independent multicentric longitudinal cohorts of patients with early multiple sclerosis (n = 309 discovery and n = 232 validation), we were able to identify three distinct peripheral blood immunological endophenotypes by a combination of high-dimensional flow cytometry and serum proteomics, followed by unsupervised clustering. Longitudinal clinical and paraclinical follow-up data collected for the cohorts revealed that these endophenotypes were associated with disease trajectories of inflammation versus early structural damage. Investigating the capacity of immunotherapies to normalize endophenotype-specific immune signatures revealed discrete effect sizes as illustrated by the limited effect of interferon-ß on endophenotype 3-related immune signatures. Accordingly, patients who fell into endophenotype 3 subsequently treated with interferon-ß exhibited higher disease progression and MRI activity over a 4-year follow-up compared with treatment with other therapies. We therefore propose that ascertaining a patient's blood immune signature before immunomodulatory treatment initiation may facilitate prediction of clinical disease trajectories and enable personalized treatment decisions based on pathobiological principles.

Single-cell profiling reveals preferential reduction of memory B cell subsets in cladribine patients that correlates with treatment response.

Background: Cladribine is a highly effective immunotherapy that is applied in two short-term courses over 2 years and reduces relapse rate and disease progression in patients with relapsing multiple sclerosis (MS). Despite the short treatment period, cladribine has a long-lasting effect on disease activity even after recovery of lymphocyte counts, suggesting a yet undefined long-term immune modulating effect. Objectives: Our aim was to provide a more profound understanding of the detailed effects of cladribine, also with regard to the patients' therapy response. Design: We performed an open-labeled, explorative, prospective, single-arm study, in which we examined the detailed lymphocyte subset development of MS patients who received cladribine treatment over 2 years. Methods: We performed in-depth profiling of the effects of cladribine on peripheral blood lymphocytes by flow cytometry, bulk RNA sequencing of sorted CD4+ T cells, CD8+ T cells, and CD19+ B cells as well as single-cell RNA sequencing of peripheral blood mononuclear cells in a total of 23 MS patients before and at different time points up to 24 months after cladribine treatment. Data were correlated with clinical and cranial magnetic resonance imaging (MRI) disease activity. Results: Flow cytometry revealed a predominant and sustained reduction of memory B cells compared to other B cell subsets after cladribine treatment, whereas T cell subsets were slightly reduced in a more uniform pattern. The overall transcriptional profile of total blood B cells exhibited reduced expression of proinflammatory and T cell activating genes, while single-cell transcriptomics revealed that gene expression within each B cell cluster did not change over time. Stable patients displayed stronger reductions of selected memory B cell clusters as compared to patients with clinical or cerebral MRI disease activity. Conclusion: We describe a pronounced and sustained effect of cladribine on the memory B cell compartment, and the resulting change in B cell subset composition causes a significant alteration of B cell transcriptional profiles resulting in reduced proinflammatory and T cell activating capacities. The extent of reduction in selected memory B cell clusters by cladribine may predict treatment response.

Learning CNS immunopathology from therapeutic interventions.

Modulation of immune cell trafficking across the blood-brain barrier has not only introduced a therapeutic avenue for multiple sclerosis (MS) but also represents an example of reverse translational medicine. Data from clinical trials of drugs such as natalizumab and fingolimod have revealed the involvement of different compartments in relapsing versus non-relapsing MS immune biology, contributed to our understanding of central nervous system (CNS) immune surveillance, and stimulated new fields of research. Here, we discuss the results of these trials, as well as patient biomaterial-based scientific projects, and how both have informed our understanding of CNS immunopathology.

Enhanced pathogenicity of Th17 cells due to natalizumab treatment: Implications for MS disease rebound.

After natalizumab (NAT) cessation, some multiple sclerosis (MS) patients experience a severe disease rebound. The rebound pathophysiology is still unclear; however, it has been linked to interleukin-17-producing T-helper (Th17) cells. We demonstrate that during NAT treatment, MCAM+CCR6+Th17 cells gradually acquire a pathogenic profile, including proinflammatory cytokine production, pathogenic transcriptional signatures, brain endothelial barrier impairment, and oligodendrocyte damage via induction of apoptotic pathways. This is accompanied by an increase in Th17 cell frequencies in the cerebrospinal fluid of NAT-treated patients. Notably, Th17 cells derived from NAT-treated patients, who later developed a disease rebound upon treatment cessation, displayed a distinct transcriptional pathogenicity profile associated with altered migratory properties. Accordingly, increased brain infiltration of patient Th17 cells was illustrated in a humanized mouse model and brain histology from a rebound patient. Therefore, peripheral blood-accumulated MCAM+CCR6+Th17 cells might be involved in rebound pathophysiology, and monitoring of changes in Th17 cell pathogenicity in patients before/during NAT treatment cessation might enable rebound risk assessment in the future.

A single-cell analysis framework allows for characterization of CSF leukocytes and their tissue of origin in multiple sclerosis.

Peripheral central nervous system (CNS)-infiltrating lymphocytes are a hallmark of relapsing-remitting multiple sclerosis. Tissue-resident memory T cells (TRM) not only populate the healthy CNS parenchyma but also are suspected to contribute to multiple sclerosis pathology. Because cerebrospinal fluid (CSF), unlike CNS parenchyma, is accessible for diagnostics, we evaluated whether human CSF, apart from infiltrating cells, also contains TRM cells and CNS-resident myeloid cells draining from the parenchyma or border tissues. Using deep generative models, we integrated 41 CSF and 14 CNS parenchyma single-cell RNA sequencing (scRNAseq) samples from eight independent studies, encompassing 120,629 cells. By comparing CSF immune cells collected during multiple sclerosis relapse with cells collected during therapeutic very late antigen-4 blockade, we could identify immune subsets with tissue provenance across multiple lineages, including CNS border-associated macrophages, CD8 and CD4 TRM cells, and tissue-resident natural killer cells. All lymphocytic CNS-resident cells shared expression of CXCR6 but showed differential ITGAE expression (encoding CD103). A common signature defined CD4 and CD8 TRM cells by expression of ZFP36L2, DUSP1, and ID2. We further developed a user interface-driven application based on this analysis framework for atlas-level cell identity transfer onto new CSF scRNAseq data. Together, these results define CNS-resident immune cells involved in multiple sclerosis pathology that can be detected and monitored in CSF. Targeting these cell populations might be promising to modulate immunopathology in progressive multiple sclerosis and other neuroinflammatory diseases.

Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis.

Epstein-Barr virus (EBV) infection precedes multiple sclerosis (MS) pathology and cross-reactive antibodies might link EBV infection to CNS autoimmunity. As an altered anti-EBV T cell reaction was suggested in MS, we queried peripheral blood T cell receptor ß chain (TCRß) repertoires of 1,395 MS patients, 887 controls, and 35 monozygotic, MS-discordant twin pairs for multimer-confirmed, viral antigen-specific TCRß sequences. We detected more MHC-I-restricted EBV-specific TCRß sequences in MS patients. Differences in genetics or upbringing could be excluded by validation in monozygotic twin pairs discordant for MS. Anti-VLA-4 treatment amplified this observation, while interferon ß- or anti-CD20 treatment did not modulate EBV-specific T cell occurrence. In healthy individuals, EBV-specific CD8+ T cells were of an effector-memory phenotype in peripheral blood and cerebrospinal fluid. In MS patients, cerebrospinal fluid also contained EBV-specific central-memory CD8+ T cells, suggesting recent priming. Therefore, MS is not only preceded by EBV infection, but also associated with broader EBV-specific TCR repertoires, consistent with an ongoing anti-EBV immune reaction in MS.

Increased Intrathecal Activity of Follicular Helper T Cells in Patients With Relapsing-Remitting Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Follicular helper T (Tfh) cells play a critical role in protective immunity helping B cells produce antibodies against foreign pathogens and are likely implicated in the pathogenesis of various autoimmune diseases. The purpose of this study was to investigate the role of Tfh cells in the pathogenesis of multiple sclerosis (MS). METHODS: Using flow cytometry, we investigated phenotype, prevalence, and function of Tfh cells in blood and CSF from controls and patients with relapsing-remitting MS (RRMS) and primary progressive MS (PPMS). In addition, an in vitro blood-brain barrier coculture assay of primary human astrocytes and brain microvascular endothelial cells grown in a Boyden chamber was used to assess the migratory capacity of peripheral Tfh cells. RESULTS: This study identified 2 phenotypically and functionally distinct Tfh cell populations: CD25- Tfh cells (Tfh1-like) and CD25int Tfh cells (Tfh17-like). Whereas minor differences in Tfh cell populations were found in blood between patients with MS and controls, we observed an increased frequency of CD25- Tfh cells in CSF of patients with RRMS and PPMS and CD25int Tfh cells in patients with RRMS, compared with controls. Increasing frequencies of CSF CD25- Tfh cells and the CD25- Tfh/Tfr ratio scaled with increasing IgG index in patients with RRMS. Despite an increased prevalence of intrathecal Tfh cells in patients with MS, no difference in the migratory capacity of circulating Tfh cells was observed between controls and patients with MS. Instead, CSF concentrations of CXCL13 scaled with total counts of Tfh and Tfr cell subsets in the CSF. DISCUSSION: Our study indicates substantial changes in intrathecal Tfh dynamics, particularly in patients with RRMS, and suggests that the intrathecal inflammatory environment in patients with RRMS promotes recruitment of peripheral Tfh cells rather than the Tfh cells having an increased capacity to migrate to CNS.

Alemtuzumab-induced immune phenotype and repertoire changes: implications for secondary autoimmunity.

Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing-remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points. Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease. In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.

Data Sharing and Reuse: A Method by the AIRR Community.

High-throughput sequencing of adaptive immune receptor repertoires (AIRR, i.e., IG and TR ) has revolutionized the ability to study the adaptive immune response via large-scale experiments. Since 2009, AIRR sequencing (AIRR-seq) has been widely applied to survey the immune state of individuals (see "The AIRR Community Guide to Repertoire Analysis" chapter for details). One of the goals of the AIRR Community is to make the resulting AIRR-seq data FAIR (Findable, Accessible, Interoperable, and Reusable) (Wilkinson et al. Sci Data 3:1-9, 2016), with a primary goal of making it easy for the research community to reuse AIRR-seq data (Breden et al. Front Immunol 8:1418, 2017; Scott and Breden. Curr Opin Syst Biol 24:71-77, 2020). The basis for this is the MiAIRR data standard (Rubelt et al. Nat Immunol 18:1274-1278, 2017). For long-term preservation, it is recommended that researchers store their sequence read data in an INSDC repository. At the same time, the AIRR Community has established the AIRR Data Commons (Christley et al. Front Big Data 3:22, 2020), a distributed set of AIRR-compliant repositories that store the critically important annotated AIRR-seq data based on the MiAIRR standard, making the data findable, interoperable, and, because the data are annotated, more valuable in its reuse. Here, we build on the other AIRR Community chapters and illustrate how these principles and standards can be incorporated into AIRR-seq data analysis workflows. We discuss the importance of careful curation of metadata to ensure reproducibility and facilitate data sharing and reuse, and we illustrate how data can be shared via the AIRR Data Commons.

Progressive multifocal leukoencephalopathy genetic risk variants for pharmacovigilance of immunosuppressant therapies.

Background: Progressive multifocal leukoencephalopathy (PML) is a rare and often lethal brain disorder caused by the common, typically benign polyomavirus 2, also known as JC virus (JCV). In a small percentage of immunosuppressed individuals, JCV is reactivated and infects the brain, causing devastating neurological defects. A wide range of immunosuppressed groups can develop PML, such as patients with: HIV/AIDS, hematological malignancies (e.g., leukemias, lymphomas, and multiple myeloma), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis, and systemic lupus erythematosus), and organ transplants. In some patients, iatrogenic (i.e., drug-induced) PML occurs as a serious adverse event from exposure to immunosuppressant therapies used to treat their disease (e.g., hematological malignancies and multiple sclerosis). While JCV infection and immunosuppression are necessary, they are not sufficient to cause PML. Methods: We hypothesized that patients may also have a genetic susceptibility from the presence of rare deleterious genetic variants in immune-relevant genes (e.g., those that cause inborn errors of immunity). In our prior genetic study of 184 PML cases, we discovered 19 candidate PML risk variants. In the current study of another 152 cases, we validated 4 of 19 variants in both population controls (gnomAD 3.1) and matched controls (JCV+ multiple sclerosis patients on a PML-linked drug ≥ 2 years). Results: The four variants, found in immune system genes with strong biological links, are: C8B, 1-57409459-C-A, rs139498867; LY9 (alias SLAMF3), 1-160769595-AG-A, rs763811636; FCN2, 9-137779251-G-A, rs76267164; STXBP2, 19-7712287-G-C, rs35490401. Carriers of any one of these variants are shown to be at high risk of PML when drug-exposed PML cases are compared to drug-exposed matched controls: P value = 3.50E-06, OR = 8.7 [3.7-20.6]. Measures of clinical validity and utility compare favorably to other genetic risk tests, such as BRCA1 and BRCA2 screening for breast cancer risk and HLA-B*15:02 pharmacogenetic screening for pharmacovigilance of carbamazepine to prevent Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis. Conclusion: For the first time, a PML genetic risk test can be implemented for screening patients taking or considering treatment with a PML-linked drug in order to decrease the incidence of PML and enable safer use of highly effective therapies used to treat their underlying disease.

Dimethyl fumarate treatment restrains the antioxidative capacity of T cells to control autoimmunity.

Dimethyl fumarate, an approved treatment for relapsing-remitting multiple sclerosis, exerts pleiotropic effects on immune cells as well as CNS resident cells. Here, we show that dimethyl fumarate exerts a profound alteration of the metabolic profile of human CD4+ as well as CD8+ T cells and restricts their antioxidative capacities by decreasing intracellular levels of the reactive oxygen species scavenger glutathione. This causes an increase in mitochondrial reactive oxygen species levels accompanied by an enhanced mitochondrial stress response, ultimately leading to impaired mitochondrial function. Enhanced mitochondrial reactive oxygen species levels not only result in enhanced T-cell apoptosis in vitro as well as in dimethyl fumarate-treated patients, but are key for the well-known immunomodulatory effects of dimethyl fumarate both in vitro and in an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis. Indeed, dimethyl fumarate immune-modulatory effects on T cells were completely abrogated by pharmacological interference of mitochondrial reactive oxygen species production. These data shed new light on dimethyl fumarate as bona fide immune-metabolic drug that targets the intracellular stress response in activated T cells, thereby restricting mitochondrial function and energetic capacity, providing novel insight into the role of oxidative stress in modulating cellular immune responses and T cell-mediated autoimmunity.

Iowa Urban FEWS: Integrating Social and Biophysical Models for Exploration of Urban Food, Energy, and Water Systems.

Most people in the world live in urban areas, and their high population densities, heavy reliance on external sources of food, energy, and water, and disproportionately large waste production result in severe and cumulative negative environmental effects. Integrated study of urban areas requires a system-of-systems analytical framework that includes modeling with social and biophysical data. We describe preliminary work toward an integrated urban food-energy-water systems (FEWS) analysis using co-simulation for assessment of current and future conditions, with an emphasis on local (urban and urban-adjacent) food production. We create a framework to enable simultaneous analyses of climate dynamics, changes in land cover, built forms, energy use, and environmental outcomes associated with a set of drivers of system change related to policy, crop management, technology, social interaction, and market forces affecting food production. The ultimate goal of our research program is to enhance understanding of the urban FEWS nexus so as to improve system function and management, increase resilience, and enhance sustainability. Our approach involves data-driven co-simulation to enable coupling of disparate food, energy and water simulation models across a range of spatial and temporal scales. When complete, these models will quantify energy use and water quality outcomes for current systems, and determine if undesirable environmental effects are decreased and local food supply is increased with different configurations of socioeconomic and biophysical factors in urban and urban-adjacent areas. The effort emphasizes use of open-source simulation models and expert knowledge to guide modeling for individual and combined systems in the urban FEWS nexus.

Classification of neurological diseases using multi-dimensional CSF analysis.

Although CSF analysis routinely enables the diagnosis of neurological diseases, it is mainly used for the gross distinction between infectious, autoimmune inflammatory, and degenerative disorders of the CNS. To investigate, whether a multi-dimensional cellular blood and CSF characterization can support the diagnosis of clinically similar neurological diseases, we analysed 546 patients with autoimmune neuroinflammatory, degenerative, or vascular conditions in a cross-sectional retrospective study. By combining feature selection with dimensionality reduction and machine learning approaches we identified pan-disease parameters that were altered across all autoimmune neuroinflammatory CNS diseases and differentiated them from other neurological conditions and inter-autoimmunity classifiers that subdifferentiate variants of CNS-directed autoimmunity. Pan-disease as well as diseases-specific changes formed a continuum, reflecting clinical disease evolution. A validation cohort of 231 independent patients confirmed that combining multiple parameters into composite scores can assist the classification of neurological patients. Overall, we showed that the integrated analysis of blood and CSF parameters improves the differential diagnosis of neurological diseases, thereby facilitating early treatment decisions.

The Future of Blood Testing Is the Immunome.

It is increasingly clear that an extraordinarily diverse range of clinically important conditions-including infections, vaccinations, autoimmune diseases, transplants, transfusion reactions, aging, and cancers-leave telltale signatures in the millions of V(D)J-rearranged antibody and T cell receptor [TR per the Human Genome Organization (HUGO) nomenclature but more commonly known as TCR] genes collectively expressed by a person's B cells (antibodies) and T cells. We refer to these as the immunome. Because of its diversity and complexity, the immunome provides singular opportunities for advancing personalized medicine by serving as the substrate for a highly multiplexed, near-universal blood test. Here we discuss some of these opportunities, the current state of immunome-based diagnostics, and highlight some of the challenges involved. We conclude with a call to clinicians, researchers, and others to join efforts with the Adaptive Immune Receptor Repertoire Community (AIRR-C) to realize the diagnostic potential of the immunome.

Sunlight exposure exerts immunomodulatory effects to reduce multiple sclerosis severity.

Multiple sclerosis (MS) disease risk is associated with reduced sun-exposure. This study assessed the relationship between measures of sun exposure (vitamin D [vitD], latitude) and MS severity in the setting of two multicenter cohort studies (nNationMS = 946, nBIONAT = 990). Additionally, effect-modification by medication and photosensitivity-associated MC1R variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-ß-treated patients. In carriers of MC1R:rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests beneficial effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS.

High anti-JCPyV serum titers coincide with high CSF cell counts in RRMS patients.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) can in rare cases occur in natalizumab-treated patients with high serum anti-JCPyV antibodies, hypothetically due to excessive blockade of immune cell migration. OBJECTIVE: Immune cell recruitment to the central nervous system (CNS) was assessed in relapsing-remitting multiple sclerosis (RRMS) patients stratified by low versus high anti-JCPyV antibody titers as indicator for PML risk. METHODS: Cerebrospinal fluid (CSF) cell counts of 145 RRMS patients were quantified by flow cytometry. Generalized linear models were employed to assess influence of age, sex, disease duration, Expanded Disability Status Scale (EDSS), clinical/radiological activity, current steroid or natalizumab treatment, as well as anti-JCPyV serology on CSF cell subset counts. RESULTS: While clinical/radiological activity was associated with increased CD4, natural killer (NK), B and plasma cell counts, natalizumab therapy reduced all subpopulations except monocytes. With and without natalizumab therapy, patients with high anti-JCPyV serum titers presented with increased CSF T-cell counts compared to patients with low anti-JCPyV serum titers. In contrast, PML patients assessed before (n = 2) or at diagnosis (n = 5) presented with comparably low CD8 and B-cell counts, which increased after plasma exchange (n = 4). CONCLUSION: High anti-JCPyV indices, which could be indicative of increased viral activity, are associated with elevated immune cell recruitment to the CNS. Its excessive impairment in conjunction with viral activity could predispose for PML development.

MCAM/CD146 Signaling via PLCγ1 Leads to Activation of ß1-Integrins in Memory T-Cells Resulting in Increased Brain Infiltration.

Multiple sclerosis is a chronic auto-inflammatory disease of the central nervous system affecting patients worldwide. Neuroinflammation in multiple sclerosis is mainly driven by peripheral immune cells which invade the central nervous system and cause neurodegenerative inflammation. To enter the target tissue, immune cells have to overcome the endothelium and transmigrate into the tissue. Numerous molecules mediate this process and, as they determine the tissue invasiveness of immune cells, display great therapeutic potential. Melanoma cell adhesion molecule (MCAM) is a membrane-anchored glycoprotein expressed by a subset of T-cells and MCAM+ T-cells have been shown to contribute to neuroinflammation in multiple sclerosis. The role of the MCAM molecule for brain invasion, however, remained largely unknown. In order to investigate the role of the MCAM molecule on T-cells, we used different in vitro and in vivo assays, including ex vivo flow chambers, biochemistry and microscopy experiments of the mouse brain. We demonstrate that MCAM directly mediates adhesion and that the engagement of MCAM induces intracellular signaling leading to ß1-integrin activation on human T-cells. Furthermore, we show that MCAM engagement triggers the phosphorylation of PLCγ1 which is required for integrin activation and thus amplification of the cellular adhesive potential. To confirm the physiological relevance of our findings in vivo, we demonstrate that MCAM plays an important role in T-cell recruitment into the mouse brain. In conclusion, our data demonstrate that MCAM expressed on T-cells acts as an adhesion molecule and a signaling receptor that may trigger ß1-integrin activation via PLCγ1 upon engagement.