Human stem cell-derived neurons and astrocytes to detect novel auto-reactive IgG response in immune-mediated neurological diseases.

Background and Objectives: 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).
Methods: 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.
Results: 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's exact test, p = 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.
Conclusion: 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.
Competing Interests: RBV received travel grants from Roche and received speaker honoraria from Novartis. None were related to this work. AH has served as an expert in advisory boards from Novocure and Bayer and received speaker honoraria from Novocure, all paid to the institution and not related to this work. AZ has patents submitted for Tensacin-R IgG, PDE10A-IgG, and DACH1-IgG as biomarkers of neurological autoimmunity, receives research funding from Roche/Genetech non-relevant to this work, and has consulted for Alexion Pharmaceutical without personal compensation. MT has served as an expert in advisory boards for Biogen, Genzyme-Sanofi, Merck, Novartis, and Roche; received travel grants from Biogen, Genzyme-Sanofi, Merck, Novartis, and Roche; and received speaker honoraria from Biogen, Novartis, and Merck. None were related to this work. CP has served as an expert in advisory boards and received travel grants from Biogen, Genzyme-Sanofi, Merck, Novartis, and Roche. None were related to this work. RDP has served on scientific advisory boards for Biogen, BMS, Merck, Novartis, Roche, and Sanofi-Genzyme and has received funding for travel or speaker honoraria from Biogen, Merck, Roche, and Sanofi-Genzyme. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2024 Mathias, Perriot, Jones, Canales, Bernard-Valnet, Gimenez, Torcida, Oberholster, Hottinger, Zekeridou, Theaudin, Pot and Du Pasquier.)