CSF single-cell RNA sequencing reveals clonally expanded CD4+ stem cell-like memory T cells in GAD65-antibody associated neurological syndromes

Barman, S.; Räuber, S.; Eisenhut, K.; Esser, D.; van Duijn, M.; Scharf, M.; Herrera-Rivero, M.; Disse, P.; Jonas, M.; Pul, D.; Heming, M.; Müller-Miny, L.; Paunovic, M.; Strippel, C.; Haholu, E.; Kaufmann, E.; Dargvainiene, J.; Kahl, S.; Ringelstein, M.; Bindels, E.; Wiendl, H.; Stoecklein, N. H.; Fischer, J.; Goebels, N.; Komorowski, L.; Roden, M.; Rossi, A.; Stoll, M.; Meuth, S. G.; Titulaer, M. J.; Leypoldt, F.; Meyer zu Hörste, G.; Thaler, F.; Melzer, N.

doi:10.1101/2025.05.18.654720

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bioRxiv · 10.1101/2025.05.18.654720

CSF single-cell RNA sequencing reveals clonally expanded CD4+ stem cell-like memory T cells in GAD65-antibody associated neurological syndromes

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Abstract

BackgroundGlutamic acid decarboxylase (GAD) antibody-associated autoimmune neurological syndromes (AINS) are a spectrum of autoimmune-mediated CNS disorders. While antibodies targeting the 65 kDa isoform of GAD are of high diagnostic value, T cell mediated cytotoxicity has been identified as a key component of disease pathogenesis. The precise pathophysiological mechanisms by which the disease is triggered and maintained, however, remain incompletely understood. MethodsWe performed single-cell transcriptome and immune repertoire sequencing (sc-seq) in CSF and blood of 8 anti-GAD65 AINS patients compared to 8 non-inflammatory controls. Monoclonal antibodies (mAbs) were synthesized from B cell receptor (BCR) data to evaluate the B cellular immune response. FindingsWe identified an increase and expansion of activated CD4+ stem cell-like memory T cells (TSCM) in the CSF of anti-GAD65 AINS patients. Expanded T cells showed increased expression of proinflammatory genes. The mAb analysis revealed a high frequency of GAD65-reactive BCRs in the CSF of anti-GAD65 AINS patients with increased somatic hypermutations compared to non-GAD-reactive BCRs and BCRs from controls. ConclusionsSc-seq identified clonally expanded CD4+ TSCM in the CSF of anti-GAD65 AINS patients harboring cytotoxic properties likely contributing to disease pathogenesis. GAD-reactive B cells circulate in the CSF of anti-GAD65 AINS patients further supporting the concept of an antigen-specific intrathecal immune response. Future studies need to clarify the actual pathogenicity of these immune cells and the link between T and B cellular immune mechanisms in the pathogenesis of anti-GAD65 AINS. FundingGerman Research Foundation (ERARE18-202 UltraAIE), German Federal Ministry of Education and Research (CONNECT GENERATE (2.0); 01GM1908A and 01GM2208A).

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Barman, S., Räuber, S., Eisenhut, K., Esser, D., van Duijn, M., Scharf, M., Herrera-Rivero, M., Disse, P., Jonas, M., Pul, D., Heming, M., Müller-Miny, L., Paunovic, M., Strippel, C., Haholu, E., Kaufmann, E., Dargvainiene, J., Kahl, S., Ringelstein, M., Bindels, E., Wiendl, H., Stoecklein, N. H., Fischer, J., Goebels, N., Komorowski, L., Roden, M., Rossi, A., Stoll, M., Meuth, S. G., Titulaer, M. J., Leypoldt, F., Meyer zu Hörste, G., Thaler, F., Melzer, N.. 2025-05-22. CSF single-cell RNA sequencing reveals clonally expanded CD4+ stem cell-like memory T cells in GAD65-antibody associated neurological syndromes. https://doi.org/10.1101/2025.05.18.654720

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