Fig. 3: Splenic CD4⁺ T-cell rearrangements in HFD-fed 5xFAD mice.

a–c CD4⁺ T-cell immune deviations in HFD-fed 5xFAD mice. Flow cytometric quantification of splenic frequencies of CD4⁺ naive T cells (CD44^lowCD62L^high; a), CD4⁺ TEMs (CD44^highCD62L^low; b), and CD4⁺FOXP3⁺ Tregs (c). Mice from two independent experiments, sample n: WT CD = 16, WT HFD = 19, 5xFAD CD = 18, 5xFAD HFD = 18. Statistical analyses: two-way ANOVA followed by Fisher’s LSD post hoc test. d–f Characterization of the CD4⁺ TEM compartment by mass cytometry. Cryopreserved splenocytes from mice evaluated for both cognition (NOR test; Fig. 1c) and systemic immune phenotype (Fig. 3a–c) were used for CyTOF analysis of the CD4⁺ TEM compartment, sample n: WT CD = 5, WT HFD = 5, 5xFAD CD = 5, 5xFAD HFD = 5. d UMAP embedding of CD4⁺ TEM cell clusters (2000 cells, randomly selected from each animal). FlowSOM-based immune cell populations are overlaid as a color dimension. e Mean population expression levels of markers used for UMAP visualization and FlowSOM clustering of CD4⁺ TEMs. f Increased frequency of exhausted TEMs in HFD-fed 5xFAD mice. Sub-clustering analysis of the CD4⁺ TEM compartment identified six clusters; Cluster 6 only is shown here, Clusters 1 to 5 are shown in Supplementary Fig. 9a. Statistical analyses: two-way ANOVA followed by Fisher’s LSD post hoc test. a–c, f Box plots represent the minimum and maximum values (whiskers), the first and third quartiles (box boundaries), the median (box internal line), and the mean (cross). Source data are provided as a Source Data file.