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Immunol."],"abstract":"<jats:sec><jats:title>Background and objectives<\/jats:title><jats:p>Up to 46% of patients with presumed autoimmune limbic encephalitis are seronegative for all currently known central nervous system (CNS) antigens. We developed a cell-based assay (CBA) to screen for novel neural antibodies in serum and cerebrospinal fluid (CSF) using neurons and astrocytes derived from human-induced pluripotent stem cells (hiPSCs).<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Human iPSC-derived astrocytes or neurons were incubated with serum\/CSF from 99 patients [42 with inflammatory neurological diseases (IND) and 57 with non-IND (NIND)]. The IND group included 11 patients with previously established neural antibodies, six with seronegative neuromyelitis optica spectrum disorder (NMOSD), 12 with suspected autoimmune encephalitis\/paraneoplastic syndrome (AIE\/PNS), and 13 with other IND (OIND). IgG binding to fixed CNS cells was detected using fluorescently-labeled antibodies and analyzed through automated fluorescence measures. IgG neuronal\/astrocyte reactivity was further analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were used as CNS-irrelevant control target cells. Reactivity profile was defined as positive using a Robust regression and Outlier removal test with a false discovery rate at 10% following each individual readout.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Using our CBA, we detected antibodies recognizing hiPSC-derived neural cells in 19\/99 subjects. Antibodies bound specifically to astrocytes in nine cases, to neurons in eight cases, and to both cell types in two cases, as confirmed by microscopy single-cell analyses. Highlighting the significance of our comprehensive 96-well CBA assay, neural-specific antibody binding was more frequent in IND (15 of 42) than in NIND patients (4 of 57) (Fisher\u2019s exact test, <jats:italic>p<\/jats:italic> = 0.0005). Two of four AQP4+ NMO and four of seven definite AIE\/PNS with intracellular-reactive antibodies [1 GFAP astrocytopathy, 2 Hu+, 1 Ri+ AIE\/PNS)], as identified in diagnostic laboratories, were also positive with our CBA. Most interestingly, we showed antibody-reactivity in two of six seronegative NMOSD, six of 12 probable AIE\/PNS, and one of 13 OIND. Flow cytometry using hiPSC-derived CNS cells or PBMC-detected antibody binding in 13 versus zero patients, respectively, establishing the specificity of the detected antibodies for neural tissue.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>Our unique hiPSC-based CBA allows for the testing of novel neuron-\/astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in established routine laboratories, opening new perspectives in establishing a diagnosis of such complex diseases.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2024.1419712","type":"journal-article","created":{"date-parts":[[2024,7,24]],"date-time":"2024-07-24T04:50:33Z","timestamp":1721796633000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Human stem cell\u2013derived neurons and astrocytes to detect novel auto-reactive IgG response in immune-mediated 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