Early adaptive immune activation detected in monozygotic twins with prodromal multiple sclerosis.
ABSTRACT: Multiple sclerosis (MS) is a disabling disease of the CNS. Inflammatory features of MS include lymphocyte accumulations in the CNS and cerebrospinal fluid (CSF). The preclinical events leading to established MS are still enigmatic. Here we compared gene expression patterns of CSF cells from MS-discordant monozygotic twin pairs. Six "healthy" co-twins, who carry a maximal familial risk for developing MS, showed subclinical neuroinflammation (SCNI) with small MRI lesions. Four of these subjects had oligoclonal bands (OCBs). By single-cell RNA sequencing of 2752 CSF cells, we identified clonally expanded CD8+ T cells, plasmablasts, and, to a lesser extent, CD4+ T cells not only from MS patients but also from subjects with SCNI. In contrast to nonexpanded T cells, clonally expanded T cells showed characteristics of activated tissue-resident memory T (TRM) cells. The TRM-like phenotype was detectable already in cells from SCNI subjects but more pronounced in cells from patients with definite MS. Expanded plasmablast clones were detected only in MS and SCNI subjects with OCBs. Our data provide evidence for very early concomitant activation of 3 components of the adaptive immune system in MS, with a notable contribution of clonally expanded TRM-like CD8+ cells.
Project description:<h4>Objective</h4>To determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients.<h4>Methods</h4>Mass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients.<h4>Results</h4>We find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood-brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related.<h4>Interpretation</h4>Our data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier.
Project description:Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.
Project description:T cells provide critical immune surveillance to the central nervous system (CNS), and the cerebrospinal fluid (CSF) is thought to be a main route for their entry. Further characterization of the state of T cells in the CSF in healthy individuals is important for understanding how T cells provide protective immune surveillance without damaging the delicate environment of the CNS and providing tissue-specific context for understanding immune dysfunction in neuroinflammatory disease. Here, we have profiled T cells in the CSF of healthy human donors and have identified signatures related to cytotoxic capacity and tissue adaptation that are further exemplified in clonally expanded CSF T cells. By comparing profiles of clonally expanded T cells obtained from the CSF of patients with multiple sclerosis (MS) and healthy donors, we report that clonally expanded T cells from the CSF of patients with MS have heightened expression of genes related to T cell activation and cytotoxicity.
Project description:<h4>Objective</h4>In multiple sclerosis (MS), central nervous system (CNS), cerebrospinal fluid (CSF), and blood display TCR clonal expansions of CD8(+) T cells. These clones have been assumed - but never demonstrated - to be similar in the three compartments. Addressing this key question is essential to infer the implication of peripheral clonally expanded CD8(+) T cells in the disease.<h4>Methods</h4>For the first time, TCR V? repertoire from paired blood (purified CD8(+) and CD4(+) T cells), CSF and CNS (22 lesions, various inflammatory and demyelination statuses) samples from three MS patients was studied using complementary determining region 3 (CDR3) spectratyping and high-throughput sequencing. In parallel, blood and CNS clonally expanded CD8(+) T cells were characterized by fluorescent staining.<h4>Results</h4>TCR V? repertoire analysis revealed strong sharing of predominant T-cell clones between CNS lesions, CSF, and blood CD8(+) T cells. In parallel, we showed that blood oligoclonal CD8(+) T cells exhibit characteristics of pathogenic cells, as they displayed a bias toward a memory phenotype in MS patients, with increased expression of CCR5, CD11a and Granzyme B (GZM-B) compared to non oligoclonal counterparts. CNS-infiltrating T cells were mainly CD8 expressing CD11a and GZM-B.<h4>Interpretation</h4>This study highlights the predominant implication of CD8(+) T cells in MS pathophysiology and demonstrates that potentially aggressive CD8(+) T cells can be easily identified and characterized from blood and CSF samples.
Project description:Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; <i>n</i> = 12), other neurologic diseases (ONDs; <i>n</i> = 1), and healthy controls (HCs; <i>n</i> = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (<i>n</i> = 4), clinically isolated syndrome (<i>n</i> = 2), and OND (<i>n</i> = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; <i>n</i> = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-?B) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-?1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.
Project description:Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune-mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF), and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS-resident B cells encounter antigen and experience affinity maturation. Paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clones were more often found in the draining CLNs. More mature clonal members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier, with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or affect the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.
Project description:We employed 19 recombinant antibodies (rAbs) generated from clonally expanded plasma cells, and native IgG from cerebrospinal fluid (CSF) of three multiple sclerosis (MS) patients for panning with phage displayed random peptide libraries. Specific peptide epitopes/mimotopes were identified and characterized. Importantly, peptide-antibody interactions were shared by rAbs and native IgG from the same patient. Three peptides strongly interacted with at least one other MS CSF, but not to inflammatory CNS controls. Database searches revealed several protein candidates including stress proteins, cell surface proteins, and neuronal proteins. Peptides derived from the candidate proteins were recognized by rAbs. Identification of peptide epitopes/mimotopes in MS may provide clues regarding disease-relevant antigens.
Project description:B cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B cells exist in the cerebrospinal fluid (CSF), meninges, and CNS parenchyma of MS patients. We sought to investigate the presence of clonally related B cells over time by performing Ig heavy chain variable region repertoire sequencing on B cells from longitudinally collected blood and CSF samples of MS patients (n = 10). All patients were untreated at the time of the initial sampling; the majority (n = 7) were treated with immune-modulating therapies 1.2 (±0.3 SD) years later during the second sampling. We found clonal persistence of B cells in the CSF of 5 patients; these B cells were frequently Ig class-switched and CD27+. Specific blood B cell subsets appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B cell persistence in CSF and blood, even in patients on immune-modulating therapy. Our findings support the concept that peripheral B cell activation and CNS-compartmentalized immune mechanisms can in part be therapy resistant.
Project description:BACKGROUND:Two human herpesviruses, human herpesvirus 6 (HHV-6), and Epstein-Barr virus (EBV), have been repeatedly linked to multiple sclerosis (MS). OBJECTIVE:The aim of this study was to investigate HHV-6 and EBV reactive oligoclonal bands (OCBs), and viral DNA in the intrathecal compartment in MS. METHODS:The reactivity of OCBs in cerebrospinal fluid (CSF) for EBV and HHV-6 antigens and stability of virus reactive OCBs over time were studied in a well-characterized MS patient cohort. Associations between virus reactive OCBs and viral DNA in CSF (and any clinical and/or radiological findings) were investigated. RESULTS:Of patients with MS, 38% had OCBs reactive to either one of the viruses studied, compared to none in the patients with other inflammatory neurological diseases (p=0.005). The banding pattern of virus reactive OCBs remained the same over time. Furthermore, MS patients with viral DNA in CSF had more contrast enhancing lesions (CELs). CONCLUSION:The stable presence of herpesvirus reactive OCBs in CSF further strengthens the association of MS with these viruses. The finding that herpesviruses might be linked to the appearance of active lesions warrants investigation of new therapeutic strategies to treat these viruses in MS.
Project description:The central nervous system (CNS) of patients with multiple sclerosis (MS) is the site where disease pathology is evident. Damaged CNS tissue is commonly associated with immune cell infiltration. This infiltrate often includes B cells that are found in multiple locations throughout the CNS, including the cerebrospinal fluid (CSF), parenchyma, and the meninges, frequently forming tertiary lymphoid structures in the latter. Several groups, including our own, have shown that B cells from distinct locations within the MS CNS are clonally related and display the characteristics of an antigen-driven response. However, the antigen(s) driving this response have yet to be conclusively defined. To explore the antigen specificity of the MS B cell response, we produced recombinant human immunoglobulin (rIgG) from a series of expanded B cell clones that we isolated from the CNS tissue of six MS brains. The specificity of these MS-derived rIgG and control rIgG derived from non-MS tissues was then examined using multiple methodologies that included testing individual candidate antigens, screening with high-throughput antigen arrays and evaluating binding to CNS-derived cell lines. We report that while several MS-derived rIgG recognized particular antigens, including neurofilament light and a protocadherin isoform, none were unique to MS, as non-MS-derived rIgG used as controls invariably displayed similar binding specificities. We conclude that while MS CNS resident B cells display the characteristics of an antigen-driven B cell response, the antigen(s) driving this response remain at large.