Extended Data Fig. 10. Profiling MNP-2 responses to TCR and PMA/iono stimulation.
(a) RNA-based UMAP of TCR (upper row) and PMA/ionomycin (lower row) stimulations for each of the 4 pediatric donors. (b) RNA-based UMAP with CD69 gene and ADT expression in unstimulated and 4h TCR (aCD3/aCD28 beads; 0.5:1 beads per cell) and 4h PMA/iono stimulated pediatric CD8 T cells (n = 4 donors). Subsets with stimulation condition are circled. (c) GSEA analysis in naive, MNP-2, and memory CD8 T cell subsets comparing TCR stimulated versus unstimulated conditions. FDR < 0.05 was considered significant in the fgsea analysis. Dot size corresponds to the percent of genes that showed enrichment for the indicated pathway and cell type. Dot color corresponds to the normalized enrichment score (NES). (d) Pseudobulk RNA expression in naive, MNP-2, and memory CD8 T cells of IFNG and SPRY2 post-TCR or post-PMA/iono stimulation. (e) Dot plot of exhaustion and senescence-related gene and protein (ADT) expression profiles in naive, MNP-2, and memory CD8 T cells pre- and post-TCR stimulation. (f) CD27 and CD28 surface protein expression in unstimulated and TCR stimulated pediatric CD8 T cells on RNA-based UMAP. (g) Effector gene profile (CCL3, CCL4, CCL5, CSF2) and (h) co-stimulatory receptor (CD137[ADT], TNFRSF9[RNA]) expression in unstimulated and PMA (50 ng/ml) plus ionomycin (1 μg/ml) (PMA/iono) stimulated pediatric CD8 T cells on RNA-based UMAP. Pseudobulk RNA expression in naive, MNP-2, and memory CD8 T cells is shown to the right of each UMAP. (i) Frequency of MNP-2 cells in total peripheral T cells in healthy (n = 6), moderate MIS-C (n = 2), recovered moderate MIS-C (n = 1), severe MIS-C (n = 5), recovered severe MIS-C (n = 1) children from an external dataset (GSE166489)). (j) TCR alpha and beta V-J gene usage in MNP-2 cells in healthy (TRA: 153 cells, TRB: 208 cells) and recovered MIS-C (TRA: 67 cells, TRB: 84 cells) children.