Extended Data Fig. 2: Murine and human neuroectodermal cells express Il12rb2.
(a-b) Representative IHC for IL-12Rβ2+and NeuN+ cells in the steady-state C57BL/6 mouse brain (m/f; data are representative for 1 out of 3 experiments); scale bar= 200 μm. (c-e) Representative RNAscope® images of EAE C57BL/6 mouse brain. Cell markers were co-stained with probes for Il12rb1 and Il12rb2, as highlighted by arrowheads. Scale bars = 25 μm (insets) or 50 μm. (f) Immunostaining of β-gal+Olig2+ and β-gal+NeuN+ cells in EAE Il12rb2LacZ/LacZ reporter mice (n = 4, m/f; data are representative for 1 out of 3 experiments). Scale bars = 50 μm or 100 μm. Insets are enlargements of outlined regions in the original images. GCL, granule cell layer; PCL, Purkinje cell layer; sg (DG-sg), dentate gyrus, granule cell layer; po (DG-po), dentate gyrus, polymorph layer; mo (DG-mo), dentate gyrus, molecular layer. (g) snRNA-seq of n = 66,432 nuclei from white matter (WM) areas of five patients with progressive MS and three age- and sex-matched non affected, non-dementia controls (accessed at GSE180759 (ref. 13). UMAP displaying identified cell clusters, sorted by cell type (OPCs: oligodendrocyte progenitor cells). (h) Schematic illustration of MS lesion (left). IL12RB1- and IL12RB2-expression overlayed on UMAP, sorted by pathological condition (right). (i) Violin plots displaying IL12RB1 and IL12RB2 transcripts in neurons and oligodendrocytes, split by pathological condition (violin plots depict the number of counts per cell across type/site of lesions ; Wilcoxon rank sum test). (j) snRNAseq of n = 39,579 nuclei from whole MS tissue sections (n = 12) and control brain tissue (n = 9) (accessed at PRJNA544731 (ref. 14). UMAP with identified cell clusters, sorted by cell type (EN: excitatory neurons; IN: interneurons; PVALB: parvalbumin; L: layer). (k) Violin plots depicting neuronal IL12RB2 transcripts, split by pathological condition (see above; Wilcoxon rank sum test). (l) Brain sections from MS patients were stained for IL-12Rβ2 or total rabbit IgG for IHC (n = 3; data are representative for 1 out of 3 experiments); scale bar= 200 μm [normal appearing grey matter (NAGM), normal appearing WM (NAWM)]. (m) Whole cell protein lysates were prepared from dissociated mouse cerebellar neurons at DIV 14 following 5-, 10- and 15-min stimulation with IL-12 (100 ng/mL), followed by western blot for total Stat4 and pStat4 (Tyr693) (loading control: actin) (n = 3 per group per time point). (n) Fold change in pSTAT4 (relative to total Stat4) protein expression. (o) Whole cell protein lysates were prepared from mouse primary oligodendrocyte cultures at DIV 4 following 5-, 10- and 15-min stimulation with IL-12 (100 ng/mL); western blot analysis was performed as in (m) (n = 3 per group per time point). (p) Quantification of pSTAT4 (relative to total Stat4). In (n) and (p) data are shown as mean ± SD and are representative for 1 out of 3 experiments. Statistical significance was evaluated by one-way ANOVA with Bonferroni multiple comparison post-hoc test (****P < 0.0001; *P = 0.0157; *P = 0.0371, left to right).
