Human stem cell derived neurons and astrocytes to detect auto-reactive IgG in neurological diseases
Up to 46% of patients with presumed autoimmune limbic encephalitis are seronegative for all currently known 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 (hiPSC). In this study, hiPSC-derived neural cells seeded on 96-well plates (96-well CBA) were incubated with serum or CSF from 99 patients, including 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 bound to fixed CNS cells were detected using a combination of fluorescently-labelled antibodies. IgG-associated fluorescence intensity measures and microscopy observations were automated. IgG reactivity to neurons and astrocytes was further analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) 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. Using our 96-well CBA, we detected antibodies recognizing hiPSC-derived neural cells in 19/99 (19.2%) study patients. Antibodies bound specifically to astrocytes in nine cases, to neurons in eight cases and to both cell types in two cases. Microscopy single-cell analyses ascertained cellular distribution and binding specificity. Highlighting the significance of our novel 96-well CBA assay, the occurrence of CNS-specific antibody binding was more frequent in IND (15/42) than in NIND patients (4/57) (Fisher test, p=0.0005). Three of three patients with astrocyte-reactive (2 AQP4+ NMO, 1 GFAP astrocytopathy), and 3/4 with intracellular neuron-reactive antibodies (2 Hu+, 1 Ri+ AIE/PNS), as identified in validated diagnostic laboratories, were also positive with our CBA assay. Most interestingly, we showed antibody-reactivity in 2/6 seronegative NMOSD, 6/12 probable AIE/PNS, and 1/13 OIND. Flow cytometry using hiPSC-derived CNS cells or PBMC detected antibody binding in 13 versus 0 patients, respectively, establishing the specificity of the detected antibodies for neural tissue. Our unique hiPSC-based 96-well CBA allows for the screening of neuron- or astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in established routine laboratories. Such a potent tool opens new perspectives in establishing early diagnosis of Ab-mediated diseases of the CNS.