Fig. 6: Unconventional CD8⁺ T cells in children with T1D.

A–C Cells were annotated using a published RNA-seq dataset of FACS-sorted TR3-56 (CD3⁺CD56⁻), NK, CD3⁺CD56⁻, and CD8⁺ cells (GSE106082) with SingleR. A Quantification of TR3-56 annotation scores in CD8⁺ and CD4⁺ T cells from healthy donors (n = 13) and children with T1D at T0 (n = 30) or T1 (n = 29). B UMAP (from Supplementary Fig. 5A) with cells annotated as NK or TR3-56 highlighted. C Violin plots of TR3-56 annotation scores across clusters; bars at medians. D Quantification of TR3-56 annotation scores for cells from different parent clusters (only cells annotated as TR3-56 considered). UMAP highlights are illustrative; violin plots show medians. E Heatmap of marker genes shared across TR3-56 cells, column-scaled by z-score. F Module scores of Treg signature genes in main CD8⁺ clusters and TR3-56 cells. Violin plots; P-values by two-tailed Wilcoxon test. G Reclustering of CD8⁺ γδT and MAIT cells from Supplementary Fig. 19B (11,012 cells, 43 donors) after reprocessing. UMAP colored by clusters. H Heatmap of marker genes defining clusters in (G). I Percentage of cells from clusters in (G) among total unconventional CD8⁺ T cells per sample in healthy (n = 13), T1D T0 (n = 30), and T1D T1 (n = 29). J, K Flow cytometry validation of CD8⁺ TR3-56 cells (gating in Supplementary Fig. 20). J Intensity of TCRγδ and CD56 in naïve CD8⁺, non-naïve CD8⁺, NK, and CD8low T cells. Shown: representative histogram (left) and quantification across 68 samples (n = 13 healthy, n = 26 T1D T0, n = 29 T1D T1). K Frequencies of γδT (left) and CD8low (middle) T cells among CD3⁺ cells, and GZMB intensity in CD8low cells (right), across the same donors. Panels (A, I, K): P-values by two-tailed Mann–Whitney test (healthy vs. T1D T0) or two-tailed paired Mann–Whitney test (T1D T0 vs. T1). Bars = medians. AgExp – Antigen Experienced T cells, MAIT – Mucosa Associated Invariant T cells, T1D – Type 1 Diabetes Mellitus, T0 – timepoint at diagnosis, T1 – timepoint one year after diagnosis.