Fig. 4

CCR2 blockade prevents accumulation of FOXP3⁺ regulatory CD4⁺ T cells in DM-hTAU brains treated with anti-PD-L1. This experiment included four groups of DM-hTAU mice treated either with: IgG, αPD-L1, αCCR2 + αPD-L1, or αCCR2. An additional group of WT mice served as healthy controls. (A) Schematic presentation of experimental design: αCCR2 was i.p. injected to DM-hTAU mice 3 days prior (Day − 3) to αPD-L1 or IgG (Day 0), and again on days 1, 5 and 9. The brains were analyzed by CyTOF 3 days after the last αCCR2 injection (Day 12). (B) FlowSOM clustering over tSNE plot showing different immune populations. DCs- dendritic cells, BAMs- border associated macrophages. (C) Heatmap of the CyTOF data showing Z-score of mean expression levels of the different markers across distinct CD45⁺ immune cell populations. (B, C) Representative results from one of three independent experiments. (D) Quantification of CD4⁺ T cells as measured by CyTOF. The percentage of each cell population per mouse was calculated, and normalized to the control IgG group. (One-way ANOVA F_(4,20) = 4.427, *p = 0.01. Post-hoc uncorrected Fisher’s LSD multiple comparisons between DM-hTAU groups to WT: #p < 0.05. Post-hoc uncorrected Fisher’s LSD multiple comparisons between the DM-hTAU groups: **p < 0.01). n = 4–6 mice per group. Data are presented as mean ± s.e.m. (E) Flow cytometry gating strategy of Tregs in the brain of DM-hTAU mice. (F) Flow cytometry analysis of Tregs obtained from two experiments, in which each group was comprised of a pool of 10 mice. The results are presented as a ratio to the control IgG group. Data are presented as mean ± s.e.m. (G) Representative flow cytometry dot plots demonstrating negligible expression of CCR2 by the Tregs detected in the brains