Extended Data Fig. 9: Nasal anti-CD3 induced Total CD4 + T-cells ameliorate microglial response and improved behavioral outcomes at chronic TBI.

(a) Schematic representing experimental timeline of adoptive transfer experiment. Created with BioRender.com. (b) Behavioral testing of rotarod, Morris water maze, probe trial, and anxiety like behavior (measured by the open field) was assessed between WT Sham (DPBS treated, baseline), Iso-total CD4⁺, aCD3-total CD4⁺, and aCD3-FoxP3^-GFP groups. Morris water maze analyzed by two-factor repeated measures two-way ANOVA (group x time); others by one-way ANOVA with Tukey’s multiple comparisons. Data shown as mean ± SEM. (c) Visual representing an experiment where splenic CD4⁺ cells from 7 days treated TBI (FoxP3-GFP) animals were injected into untreated, but TBI-injured (CD45.1) animals and the %CD45.2 cells were analyzed by fluorescence-activated cell sorting (FACS) at 3 days after injection. Flow cytometry gating of brain, cervical lymph node, and spleen of (CD45.1) animals showing the percent of CD45.2 cell infiltration. n = 5 mice and the brain was a pool of 5 ipsilateral hemispheres. Created with BioRender.com. (d) Heatmap of DEGs from microglia 30 days following TBI and adoptive transfer identified using DESeq2 analysis (two-sided likelihood ratio test, n = 4 mice/group, FDR-corrected P < 0.05). Clusters of genes were functionally annotated using enriched GO Biological Process (BP) terms (q-value < 0.05). (e) GSEA analysis of GO Biological Process (BP) 30 days post-TBI based on the following comparisons: aCD3-Total CD4⁺ vs. Iso-Total CD4⁺, and aCD3-FoxP3^-GFP vs. Iso-total CD4⁺. Asterisk (*) indicates enriched terms (q-value < 0.05). NES, normalized enrichment score; NS, not significant. (f) Volcano plot of DEGs in aCD3-total CD4⁺ vs. aCD3-FoxP3^-GFP 30 days post-TBI identified using DESeq2 (two-sided Wald test, n = 4 mice/group). Labeled genes have an FDR-corrected P < 0.05. (g) GSEA analysis of GO Biological Process (BP) comparing aCD3-total CD4⁺ vs. aCD3-FoxP3^-GFP 30 days post-TBI. NES, normalized enrichment score. (h) Predicted top upstream regulators using IPA analysis based on DEGs in aCD3-total CD4⁺ vs. aCD3-FoxP3^-GFP 30 days post-TBI. (i) RT- qPCR of ipsilateral hemisphere 30 days post-TBI and expression normalized to GAPDH. Data shown as mean ± SEM, n = 5 mice/group and analyzed by one-way ANOVA with Tukey’s multiple comparisons. All data are biological replicates and are representative from two independent experiments. n.s. = non-significant.