Pot, C. | Search bioRxiv

bioRxiv2024

KIR3DL1 and Tox identify clonally expanded encephalitogenic neuron-specific CD8+ T cells in autoimmune encephalitis

Autoreactive CD8+ T cells are the principal suspects in autoimmune encephalitis (AIE) with antibodies targeting intracellular neuronal antigens So far, the search for neuron-autoreactive CD8+ T cells has been focused on a few autoantigens and did not yield convincing results. Here, we leveraged natural antigen presentation by hiPSC-derived neurons to look at the global autoreactive CD8+ T cell response, independently of pre-conceived hypothesis of the autoantigens involved in the disease. This unbiased approach allowed for the identification of rare polyclonal neuron-reactive CD8+ T cells in healthy donors, and contrastingly, expanded clonotypes in two patients with anti-Ri AIE. Detailed ex vivo phenotypic characterization of these clonotypes revealed a specific transcriptional program suggestive of a pathogenic potential. In particular, this subset can be identified by the expression of KIR3DL1 and TOX. Strikingly, we could also demonstrate that CD8+ T cells found in the brain of an anti-Ri AIE patient display a similar phenotype associated with cytotoxicity and encephalitogenic features.

immunology↗

bioRxiv2024

Human stem cell derived neurons and astrocytes to detect auto-reactive IgG in neurological diseases

Background and objectivesUp 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 CSF using neurons and astrocytes derived from human induced pluripotent stem cells (hiPSC). MethodsHuman 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-labelled antibodies and analyzed through automated fluorescence measures. IgG neuronal/astrocyte reactivity 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. ResultsUsing 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 novel 96-well CBA assay, neural-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 diagnostic laboratories, were also positive with our CBA. 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. DiscussionOur unique hiPSC-based CBA allows for the screening 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 early diagnosis of such complex diseases.

neuroscience↗