Cell-binding IgM in CSF is distinctive of multiple sclerosis and targets the iron transporter SCARA5.

Intrathecal IgM production in multiple sclerosis is associated with a worse disease course. To investigate pathogenic relevance of autoreactive IgM in multiple sclerosis, CSF from two independent cohorts, including multiple sclerosis patients and controls, were screened for antibody binding to induced pluripotent stem cell-derived neurons and astrocytes, and a panel of CNS-related cell lines. IgM binding to a primitive neuro-ectodermal tumour cell line discriminated 10% of multiple sclerosis donors from controls. Transcriptomes of single IgM producing CSF B cells from patients with cell-binding IgM were sequenced and used to produce recombinant monoclonal antibodies for characterization and antigen identification. We produced five cell-binding recombinant IgM antibodies, of which one, cloned from an HLA-DR + plasma-like B cell, mediated antigen-dependent complement activation. Immunoprecipitation and mass spectrometry, and biochemical and transcriptome analysis of the target cells identified the iron transport scavenger protein SCARA5 as the antigen target of this antibody. Intrathecal injection of a SCARA5 antibody led to an increased T cell infiltration in an experimental autoimmune encephalomyelitis (EAE) model. CSF IgM might contribute to CNS inflammation in multiple sclerosis by binding to cell surface antigens like SCARA5 and activating complement, or by facilitating immune cell migration into the brain.

SARS-CoV-2 infects epithelial cells of the blood-cerebrospinal fluid barrier rather than endothelial cells or pericytes of the blood-brain barrier.

BACKGROUND: As a consequence of SARS-CoV-2 infection various neurocognitive and neuropsychiatric symptoms can appear, which may persist for several months post infection. However, cell type-specific routes of brain infection and underlying mechanisms resulting in neuroglial dysfunction are not well understood. METHODS: Here, we investigated the susceptibility of cells constituting the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP) to SARS-CoV-2 infection using human induced pluripotent stem cell (hiPSC)-derived cellular models and a ChP papilloma-derived epithelial cell line as well as ChP tissue from COVID-19 patients, respectively. RESULTS: We noted a differential infectibility of hiPSC-derived brain microvascular endothelial cells (BMECs) depending on the differentiation method. Extended endothelial culture method (EECM)-BMECs characterized by a complete set of endothelial markers, good barrier properties and a mature immune phenotype were refractory to SARS-CoV-2 infection and did not exhibit an activated phenotype after prolonged SARS-CoV-2 inoculation. In contrast, defined medium method (DMM)-BMECs, characterized by a mixed endothelial and epithelial phenotype and excellent barrier properties were productively infected by SARS-CoV-2 in an ACE2-dependent manner. hiPSC-derived brain pericyte-like cells (BPLCs) lacking ACE2 expression were not susceptible to SARS-CoV-2 infection. Furthermore, the human choroid plexus papilloma-derived epithelial cell line HIBCPP, modeling the BCSFB was productively infected by SARS-CoV-2 preferentially from the basolateral side, facing the blood compartment. Assessment of ChP tissue from COVID-19 patients by RNA in situ hybridization revealed SARS-CoV-2 transcripts in ChP epithelial and ChP stromal cells. CONCLUSIONS: Our study shows that the BCSFB of the ChP rather than the BBB is susceptible to direct SARS-CoV-2 infection. Thus, neuropsychiatric symptoms because of COVID-19 may rather be associated with dysfunction of the BCSFB than the BBB. Future studies should consider a role of the ChP in underlying neuropsychiatric symptoms following SARS-CoV-2 infection.

Comprehensive proteomic analysis of JC polyomavirus-infected human astrocytes and their extracellular vesicles.

IMPORTANCE: Progressive multifocal leukoencephalopathy is a crimpling demyelinating disease of the central nervous system caused by JC polyomavirus (JCPyV). Much about JCPyV propagation in the brain remains obscure because of a lack of proper animal models to study the virus in the context of the disease, thus hampering efforts toward the development of new antiviral strategies. Here, having established a robust and representative model of JCPyV infection in human-induced pluripotent stem cell-derived astrocytes, we are able to fully characterize the effect of JCPyV on the biology of the cells and show that the proteomic signature observed for JCPyV-infected astrocytes is extended to extracellular vesicles (EVs). These data suggest that astrocyte-derived EVs found in body fluids might serve as a rich source of information relevant to JCPyV infection in the brain, opening avenues toward better understanding the pathogenesis of the virus and, ultimately, the identification of new antiviral targets.

Ocrelizumab Impairs the Phenotype and Function of Memory CD8+ T Cells: A 1-Year Longitudinal Study in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: Depleting CD20+ B cells is the primary mechanism by which ocrelizumab (OCRE) is efficient in persons with multiple sclerosis (pwMS). However, the exact role of OCRE on other immune cell subsets directly or indirectly remains elusive. The purpose of this study is to characterize the dynamics of peripheral immune cells of pwMS on OCRE. METHODS: We collected blood samples from 38 pwMS before OCRE onset (T0) and at 6 and 12 months (T6, T12) after initiation. To cover the immune cell diversity, using mass cytometry time of flight, we designed a 38-parameter panel to analyze B, T, and innate immune cell markers and CNS migratory markers. In parallel, viral-specific CD8+ T-cell responses were assessed by the quantification of interferon-γ secretion using the enzyme-linked immunospot assay on cytomegalovirus, Epstein-Barr virus, and influenza stimulations. RESULTS: Beside B-cell depletion, we observed a loss in memory CD8+CD20+ and central memory CD8+ T cells but not in CD4+CD20+ T cells already at T6 and T12 (p < 0.001). The loss of memory CD8+ T cells correlated with a lower CXCR3 expression (p < 0.001) and CNS-related LFA-1 integrin expression (p < 0.001) as well as a reduced antiviral cellular immune response observed at both time points (p < 0.001). Of note, we did not observe major changes in the phenotype of the other cell types studied. Seven of 38 (18.4%) patients in our cohort presented with infections while on OCRE; 4 of which were switched from dimethyl fumarate. Finally, using a mixed linear model on mass cytometry data, we demonstrated that the immunomodulation induced by previous disease-modifying therapies (DMTs) was prolonged over the period of the study. DISCUSSION: In addition to its well-known role on B cells, our data suggest that OCRE also acts on CD8+ T cells by depleting the memory compartment. These changes in CD8+ T cells may be an asset in the action of OCRE on MS course but might also contribute to explain the increased occurrence of infections in these patients. Finally, although more data are needed to confirm this observation, it suggests that clinicians should pay a special attention to an increased infection risk in pwMS switched from other DMTs to OCRE.

Generation of transgene-free human induced pluripotent stem cells from erythroblasts in feeder-free conditions.

This protocol describes the generation and characterization of human induced pluripotent stem cells (hiPSCs) from erythroblasts. A key difference with classical protocols is the reprogramming of erythroblasts from a simple blood draw as opposed to fibroblasts/keratinocytes, which requires a biopsy. Moreover, working with erythroblasts ensures that no recombination of the TCR/BCR genes occurs, as opposed to T cells and whole peripheral blood mononuclear cells-based approaches. Last, this approach uses non-integrative episomes ensuring no integration of transgenes into the hiPSCs genome. For complete details on the use and execution of this protocol, please refer to Perriot et al. (2018).