Novel Cell-Based Assay Enhances Detection of Autoantibodies in Neurological Disorders

Recently published in Frontiers in Immunology, a cell-based assay (CBA) developed by researchers allowed the testing of novel neuron-/astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in routine laboratories. This CBA, using neurons and astrocytes derived from human-induced pluripotent stem cells (hiPSCs), can potentially open new viewpoints in establishing a diagnosis of complex diseases such as neuromyelitis optica spectrum disorder (NMOSD).¹

Using this 96-well CBA, investigators identified antibodies that recognized hiPSC-derived neural cells in 19 out of 99 patients. Antibodies were attached particularly to astrocytes in 9 cases, to neurons in 8 cases, and to both cell types in 2 cases, which were confirmed by microscopy single-cell analyses. Investigators observed that neural-specific antibody binding was more frequent in patients with inflammatory neurological diseases (IND, n = 15) compared with those who had non-IND (NIND, n = 4) (Fisher's exact test, P = .0005), further highlighting the significance of their comprehensive CBA.

"Our CBA using human iPSC-derived neurons and astrocytes allows to detect antibodies that would not be identified by traditional approaches. Indeed, these human central nervous system (CNS) cells naturally express antigens with the appropriate post-translational changes, which is not always the case with other methods reported to date. So, in the future, we envision that this CNS CBA has its place in the cases where an autoimmune neurological condition is strongly suspected, yet traditional detection assays give negative results,” senior author Renaud Du Pasquier, MD, told NeurologyLive^®. "For instance, up to 46% of auto-immune encephalitis (AIE) and up to 16% of NMOSD are seronegative with current traditional assays. So, our CBA, by ascertaining the diagnosis of AIE or NMOSD in some patients, may decrease the area of uncertainty and thus help the physician in therapeutical management."

All told, human iPSC-derived astrocytes or neurons were incubated with serum/CSF from 42 patients with IND and 57 patients with non-IND. Researchers noted that the IND group had 11 patients with established neural antibodies prior, 6 with seronegative NMOSD, 12 with suspension of autoimmune encephalitis/paraneoplastic syndrome (PNS), and 13 with other IND. Immunoglobulin G (IgG) binding to fixed CNS cells was identified utilizing fluorescently-labeled antibodies and evaluated by automated fluorescence measures. IgG neuronal/astrocyte reactivity was further analyzed by flow cytometry. Authors used peripheral blood mononuclear cells (PBMC) as CNS-irrelevant control target cells. Researchers defined reactivity profile as positive through a Robust regression as well as an outlier removal test with a false discovery rate at 10% following each patient readout.

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Investigators also observed that 2 out of the 4 patients with aquaporin-4 antibody positive (AQP4+) NMOSD and 4 of 7 definite AIE/PNS with intracellular-reactive antibodies, as detected in diagnostic laboratories, tested also positive with the CBA. Notably, authors demonstrated antibody-reactivity in 2 out of the 6 patients who were seronegative NMOSD, 6 of 12 probable AIE/PNS, and 1 of 13 other IND. Flow cytometry utilizing hiPSC-derived CNS cells or PBMC-identified antibody binding among 13 patients versus no patients, respectively, which established the specificity of the recognized antibodies in the neural tissue.

"One of the most difficult aspects was establishing reliable protocols to generate large numbers of hiPSC-derived astrocytes and neurons. These protocols are needed to ensure consistency and high cell quality across experiments and different teams," Du Pasquier, who serves as a full professor and the head of the neurology department at CHUV/University of Lausanne, said. "After rigorous optimization, we successfully created protocols that consistently produce high-quality CNS cells. These protocols are now used by collaborators and other research teams worldwide, ensuring standardization and reliability across various labs."

"Another significant hurdle was automating the detection of antibody reactivity through fluorescence measurements including microscopy assessments. This step is crucial for determining whether autoantibodies in serum or CSF bind to astrocytes or neurons. Such optimization enables us to generate reliable results within a few days," Du Pasquier added.

Authors noted that this current CBA is limited to only astrocytes and neurons; thus, they underscored that other CNS cell types including oligodendrocytes, microglia, endothelial or even ependymal cells could be an asset to broaden the antigenic diversity. Researchers argued the previous research showed that astrocytes and neurons presently cover the majority of CNS antigens associated with neurological disorders expect for myelin oligodendrocyte glycoprotein antibody disease, which is express by oligodendrocytes.^2,3

"An advantage of our CBA is that it offers the potential to precisely characterize new antigens that would be expressed exclusively in human brain cells. Once such an antigen is defined, it will be possible to transfect it into HEK cells and implement those cells into routine diagnostic tests. Overall, by broadening the number of antigens recognized by antibodies in autoimmune neurological diseases, our CBA may improve clinical management of patients affected by these severe conditions,” Du Pasquier said.

REFERENCES
1. Mathias A, Perriot S, Jones S, et al. Human stem cell-derived neurons and astrocytes to detect novel auto-reactive IgG response in immune-mediated neurological diseases. Front Immunol. 2024;15:1419712. Published 2024 Jul 24. doi:10.3389/fimmu.2024.1419712
2. Sechi E, Flanagan EP. Antibody-Mediated Autoimmune Diseases of the CNS: Challenges and Approaches to Diagnosis and Management. Front Neurol. 2021;12:673339. Published 2021 Jul 7. doi:10.3389/fneur.2021.673339
3. Hamid SHM, Whittam D, Mutch K, et al. What proportion of AQP4-IgG-negative NMO spectrum disorder patients are MOG-IgG positive? A cross sectional study of 132 patients. J Neurol. 2017;264(10):2088-2094. doi:10.1007/s00415-017-8596-7