Extended Data Fig. 7: Nasal anti-CD3 increases expression of phagocytosis machinery at 16 hours post injection of apoptotic neurons.

(a) Schematic presenting phagocytosis functional study. Created with BioRender.com. (b) In-vivo phagocytosis functional experiment where mice were injected with labelled apoptotic neurons and sacrificed 16 h post injection. Gating strategy showing phagocytic positive microglia in TBI-Iso and TBI-aCD3 animals. Data shown as box plots (min, max, interquartile range, median) and n = 5 mice/group were used. Data was analyzed by two-sided unpaired Student’s t-test. (c) Clustered heatmap of select DEGs of aggregated samples for phagocytic ( + P) and non-phagocytic (-P) microglia at 7 days following TBI and 16 hours post-injection of apoptotic neurons using DESeq2 analysis (two-sided likelihood ratio test, n = 5-6 mice/group, FDR-corrected P < 0.05). Identified genes pertinent to microglial phagocytosis and related functions are visualized through bar plots with log2-fold changes in phagocytic TBI-aCD3 microglia compared to non-phagocytic TBI-Iso microglia. (d) GSEA analysis of GO Biological Process (BP) comparing phagocytic TBI-aCD3 microglia to phagocytic TBI-Iso microglia at 7 days following TBI and 16 hours post-injection of apoptotic neurons. NES, normalized enrichment score. (e) Bar plots of select microglial homeostatic and neurodegenerative microglia (MGnD) markers in phagocytic TBI-aCD3 microglia compared to phagocytic TBI-Iso microglia at 7 days following TBI and 16 hours post-injection of apoptotic neurons. DEGs indicated with an asterisk: FDR-corrected P < 0.05; DEGs indicated with “*P”: P < 0.05 (DESeq2 analysis, two-sided Wald test, n = 5 mice/group). (f) Gating strategy showing how phagocytic microglia cells were identified. All data are biological replicates and are representative from two independent experiments. n.s. = non-significant.