Fig. 2: Nasal aCD3 expands FoxP3 T_reg cells and modulates the adaptive immune response.

a,b, Flow cytometry analysis and quantification of CD4⁺ (a) and CD4⁺FoxP3⁺ (b) T_reg cells in the meninges and ipsilateral hemisphere at 1, 3, 7, 14 and 30 d (D) after TBI and treatment. c, Quantification of CD4⁺ subsets at the same time points. d, Analysis of CD11b⁺-infiltrated cells across these intervals. Groups included sham-iso (n = 4), TBI-iso (n = 6) and TBI-aCD3 (n = 6). Data are analyzed by one-way ANOVA with Tukey’s multiple comparisons for every individual timepoint. Data are shown as mean ± s.e.m. representing biological replicates from two independent experiments per timepoint for a–d. e, Immunofluorescence of meninges (2 d post-TBI) and brain (7 d post-TBI) samples from FoxP3-GFP mice with DAPI (blue), CD3 (pink), FoxP3 (green). Scale bars, 100 µm. IHC, immunohistochemistry. f, PCA plot of brain and blood T_reg cells 7 d post-TBI. Brain and blood samples are pools of 5 mice and sham-iso brain samples are pools of 20 mice. Due to the low number of FoxP3⁺ cells recruited to the brain and ethical considerations, we limited the study to two biological replicates, following practices from previous studies in the field²³. Despite this limitation, the consistent and robust results observed support the validity of our findings. g,h, Heatmaps of DEGs from blood (g) and brain (h) T_reg cells at 7 d post-TBI using DESeq2 (FDR-corrected P < 0.05, n = 2 pooled samples per group). i, GSEA of GOBP 7 d post-TBI for brain T_reg cells. The asterisks indicate enriched terms (q < 0.05). NES, normalized enrichment score. j, IPA analysis of DEGs from brain T_reg cells in TBI-aCD3 versus TBI-iso using DESeq2 analysis (two-sided Wald’s test, FDR-corrected P < 0.05). One-sided Fisher’s exact test was used: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Results with FDR-corrected P < 0.05 were selected. k, Predicted upstream regulators using IPA for TBI-aCD3 versus TBI-iso. l, Quantification of FoxP3⁺IL-10⁺ T_reg cells in the ipsilateral hemisphere 7 d post-TBI. Groups included sham-iso (n = 4), TBI-iso (n = 6) and TBI-aCD3 (n = 6). Data are shown as box plots (min., max., IQR, median), analyzed by one-way ANOVA with Tukey’s multiple comparisons. Data are from biological replicates and represent two independent experiments.