T cells are considered pivotal in the pathology of multiple sclerosis (MS), but their function and antigen specificity are unknown. To unravel the role of T cells in MS pathology, we performed a comprehensive analysis on T cells recovered from paired blood, cerebrospinal fluid (CSF), normal-appearing white matter (NAWM) and white matter lesions (WML) from 27 MS patients with advanced disease shortly after death. The differentiation status of T cells in these compartments was determined by ex vivo flow cytometry and immunohistochemistry. T-cell reactivity in short-term T-cell lines (TCL), generated by non-specific stimulation of T cells recovered from the same compartments, was determined by intracellular cytokine flow cytometry. Central memory T cells predominated in CSF and effector memory T cells were enriched in NAWM and WML. WML-derived CD8+ T cells represent chronically activated T cells expressing a cytotoxic effector phenotype (CD95L and granzyme B) indicative for local antigenic stimulation (CD137). The same lesions also contained higher CD8+ T-cell frequencies expressing co-inhibitory (TIM3 and PD1) and co-stimulatory (ICOS) T-cell receptors, yet no evidence for T-cell senescence (CD57) was observed. The oligoclonal T-cell receptor (TCR) repertoire, particularly among CD8+ T cells, correlated between TCL generated from anatomically separated WML of the same MS patient, but not between paired NAWM and WML. Whereas no substantial T-cell reactivity was detected towards seven candidate human MS-associated autoantigens (cMSAg), brisk CD8+ T-cell reactivity was detected in multiple WML-derived TCL towards autologous Epstein–Barr virus (EBV) infected B cells (autoBLCL). In one MS patient, the T-cell response towards autoBLCL in paired intra-lesional TCL was dominated by TCRVβ2+CD8+ T cells, which were localized in the parenchyma of the respective tissues expressing a polarized TCR and CD8 expression suggesting immunological synapse formation in situ. Collectively, the data suggest the involvement of effector memory cytotoxic T cells recognizing antigens expressed by autoBLCL, but not the assayed human cMSAg, in WML of MS patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-017-1744-4) contains supplementary material, which is available to authorized users.

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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the cause of coronavirus disease 2019 . Little is known about the interplay between pre-existing immunity towards endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in mild and severe COVID-19 patients and disease control patients. Antibody reactivity towards nucleocapsid and spike antigens was assessed and correlated to SARS-CoV-2 neutralization. COVID-19 patients mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings support a boost of poorly protective coronavirusspecific antibodies in COVID-19 patients that correlates with disease severity, revealing original antigenic sin.

Duchenne muscular dystrophy (DMD) is the most prevalent inheritable muscle disease. It is caused by mutations in the approximately 2.5-megabase dystrophin (Dys) encoding gene. Therapeutic attempts at DMD have relied on injection of allogeneic Dys-positive myoblasts. The immune rejection of these cells and their limited availability have prompted the search for alternative therapies and sources of myogenic cells. Stem cell-based gene therapy aims to restore tissue function by the transplantation of gene-corrected autologous cells. It depends on (i) the capacity of stem cells to participate in tissue regeneration and (ii) the efficient genetic correction of defective autologous stem cells. We explored the potential of bone marrow-derived human mesenchymal stem cells (hMSCs) genetically modified with the full-length Dys-coding sequence to engage in myogenesis. By tagging hMSCs with enhanced green fluorescent protein (EGFP) or the membrane dye PKH26, we demonstrated that they could participate in myotube formation when cultured together with differentiating human myoblasts. Experiments performed with EGFP-marked hMSCs and DsRed-labeled DMD myoblasts revealed that the EGFP-positive DMD myotubes were also DsRed-positive indicating that hMSCs participate in human myogenesis through cellular fusion. Finally, we showed that hMSCs transduced with a tropism-modified high-capacity hybrid viral vector encoding full-length Dys could complement the genetic defect of DMD myotubes.

Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (∼10−6 to 10−8), its practical application has been largely restricted to specific experimental systems that allow selection of the few cells that become genetically modified. HR-mediated gene targeting has nonetheless revolutionized genetics by greatly facilitating the analysis of mammalian gene function. Recent studies showed that generation of double-strand DNA breaks at specific loci by designed endonucleases greatly increases the rate of homology-directed gene repair. These findings opened new perspectives for HR-based genome editing in higher eukaryotes. Here, we demonstrate by using donor DNA templates together with the adeno-associated virus (AAV) Rep78 and Rep68 proteins that sequence- and strand-specific cleavage at a native, predefined, human locus can also greatly enhance homology-directed gene targeting. Our findings argue for the development of other strategies besides direct induction of double-strand chromosomal breaks to achieve efficient and heritable targeted genetic modification of cells and organisms. Finally, harnessing the cellular HR pathway through Rep-mediated nicking expands the range of strategies that make use of AAV elements to bring about stable genetic modification of human cells.