Fig. 2: Human iPSC-derived neurons trigger the expansion of rare neuron-reactive CD8⁺ T cell clonotypes in healthy donors.

a Schematic representation of the experimental workflow for the identification of neuron-reactive CD8⁺ T cell clonotypes. First, a coculture of PBMCs with autologous neurons is performed for 14 days. Second, TCR-β chain repertoires at day 14 of coculture are compared to ex vivo TCR-β repertoires to identify expanded TCR-β chains. Third, paired TCR-α chains of expanded clonotypes are identified by scTCRseq, and both TCR-α and TCR-β chains are co-transfected into NFAT-luciferase reporter Jurkat CD8⁺ T cells, which are then cultured overnight with neurons. Luminescence values are then measured on a multimode microplate reader (see methods). Created in BioRender. Perriot, S. (2025) https://BioRender.com/duayzpo. b TCR-β chain repertoire clonality (top) and Shannon entropy (bottom) of CD8⁺ T cells ex vivo and after 14 days of neuron-PBMC coculture for all 6 HDs (two-sided Wilcoxon tests). n = 6 biological replicates (i.e., donors), performed once per donor across 2 independent experiments. c TCR-β chain repertoire analysis of the CD8⁺ T cells from 6 HD ex vivo and after 14 days of coculture. Each slice represents a unique TCR-β sequence. Slice size represents the frequency of each sequence in comparison with the total TCR-β chain sequences. Colored slices represent TCR-β chains that underwent a 9-fold expansion as compared to ex vivo frequencies, and that constitute >0.5% of total TCR-β after 14 days of coculture. The asterisk indicates the only TCR-β representing more than 0.5% ex vivo (HD1). The value at the center of each pie chart represents the clonality of each TCR-β repertoire. All TCR-β sequences <1:1000 are grouped into one slice (black and grey patterned slice). n = 6 biological replicates (i.e., donors), performed once per donor across 2 independent experiments. d Luminescence values of TCR-transfected NFAT-luciferase Jurkat cells after overnight culture with autologous neurons. Overnight culture was performed with Jurkat cells bearing TCRs that were identified as expanded in HD4 (blue) or prevalent TCR-β chains at day 14 that were non-expanded (grey). All luminescence values are adjusted to the background signal emitted by each respective TCR. For each clonotype, experimental conditions include neurons treated and non-treated with IFNγ + TNFα (thus HLA class I positive and negative, respectively) or with the addition of a blocking anti-HLA-ABC antibody (clone W6/32). Differences between resting vs stimulated conditions were assessed by a Kruskal–Wallis test: ns, not significant; **p < 0.01; ***p < 0.001. Expanded clonotypes, +IFNγ/TNFα vs +IFNγ/TNFα/anti-HLA: adj. p value = 0.0002. Expanded clonotypes, +IFNγ/TNFα vs -IFNγ/TNFα: adj. p value = 0.0021. n = 6 biological replicates (i.e., unique TCRs), this experiment was performed once. Data are presented as mean values ± SEM.