Fig. 4: Neuron-reactive KIR⁺CD8⁺ T cells are a common feature in aged donors and Ri-AIE patients.

a Schematic representation of the experimental workflow for the identification of the neuron-reactive CD8⁺ T cell clonotypes and associated phenotypes. Single-cell RNA sequencing (scRNAseq) was performed on ex vivo circulating CD8⁺ T cells from seven patients with Ri-AIE (including Ri01) as well as three sex-and age-matched donors without an autoimmune disease (AgDs). Data were analyzed altogether by unsupervised clustering. Both neuron-reactivity (via TCR-transfection into NFAT-luciferase reporter Jurkat cells) and phenotype of selected clonotypes were further investigated in Ri-AIE vs AgD. Created in BioRender. Perriot, S. (2025) https://BioRender.com/nhxjm7k. b UMAP displaying unsupervised clustering of ex vivo CD8⁺ T cells from three AgDs and seven Ri-AIE patients assessed by scRNAseq, resulting in 16 different clusters. c Highlighting of cluster 1 (green) and CD8⁺ T cells presenting with the same TCR-β chain CDR3 amino acid sequence as the strongly expanded neuron-reactive clonotype from Ri01 (red). The pie chart on the top right highlights the cluster repartition of this clonotype, with each cluster represented in a different color and slices representing the percentage of cells belonging to each cluster from (b). d Pie charts representing the distribution of clonotypes (left) and cells (right) from AgDs (top) and Ri-AIE patients (bottom) present in cluster 1. The proportion of CD8⁺ T cells with one unique TCR is displayed in dark grey and those with ≥2 identical TCR in light grey (i.e., expanded ex vivo). Clonotypes were selected and tested for neuron-reactivity if they presented with the following criteria: (1) a clonotype with ≥10 CD8⁺ T cells in cluster 1; (2) >60% of CD8⁺ T cells from this clonotype present in cluster 1. From CD8⁺ T cells fulfilling these criteria, we selected 27 from AgD (blue slice) and 21 from Ri-AIE (orange slice). e Dot plot representing luminescence values of TCR-transfected NFAT-luciferase Jurkat cells from AgD (blue) or Ri-AIE (orange) after overnight culture with HLA-enhanced neurons (matched for HLA haplotype). Horizontal dotted line represents positivity threshold established at a luminescence value of 5000 (neuron-reactive CD8⁺ T cell clonotypes are highlighted in darker shades of blue (AgD) or orange (Ri-AIE)). For both groups, a control condition with a blocking anti-HLA-ABC was included to ensure that the activation of Jurkat cells was TCR-mediated (two-sided Wilcoxon test, AgD p value = 0.001, Ri AIE p value = 0.0003 among neuron-reactive CD8⁺ T cells). n = 27 for AgD and n = 21 for Ri-AIE, neuron-reactive clonotypes were validated once per TCR across two independent experiments. Data are presented as mean values ± SEM. f Bar plots summarizing the proportion of neuron-reactive clones (blue) vs non-reactive clones (light blue) among AgDs (left) and Ri-AIE patients (orange vs light orange, right). g Heatmap displaying differentially expressed genes among all 16 clusters (x-axis). Genes displayed on y-axis are classified into functional categories conventionally used to classify CD8⁺ T cell phenotype. Upregulated genes are displayed in red with downregulated genes in blue.