Figure 2. Reduced circulation of immune cell subsets in homeostatic blood of 6–8 month mice lacking S1PR3.

SPADE trees consisting of live, single immune cell subsets from the peripheral blood of 6–8 month mice without injury (A, C). (A) SPADE tree generated from CD11b⁺ cells (A-I, II) with a manually gated overlay of monocytes (A-I) and neutrophils (A-II) annotated in the greater CD11b⁺ tree. The SPADE nodes are visualized in grayscale whereas colored nodes represent a heatmap form of normalized cell frequency within each node of the respective phenotypic manual overlay. All nodes corresponding to a single phenotypic subset are grouped into a single colored annotation, as denoted with a phenotype key. (B) Heatmap containing all nodes from the CD11b⁺ SPADE tree in (A) in which each phenotypic subset is denoted with a colored bar including all nodes that make up that phenotype. The colored bars on the bottom of the heatmap correspond to the colored annotations in (A). (C) SPADE tree generated from CD3⁺ T cells (C-I, II) where CD4⁺ (C-I) and CD8⁺ (C-II) T cells are manually gated and overlaid onto the T cell SPADE tree. (D) Heatmap containing all nodes from the CD3⁺ SPADE tree (C) in which CD4⁺ and CD8⁺ T cells are denoted with purple and green colored bars, respectively, accompanying their annotations in (C). (E) Hemavet concentrations of various immune cell subsets circulating the peripheral blood of mice lacking S1PR3 compared to their WT controls. Notable differences are seen within the 6–8 month age range. Statistical analyses conducted using two-way ANOVA (E-I) with Tukey’s multiple comparisons and two-tailed t-tests (E-II-E-V). Data presented as mean ± S.E.M, *p< 0.05. n=3 samples (A, B) or 7 samples (C, D) per genotype for SPADE analysis. n=4 WT samples and 6 S1PR3 KO samples for Hemavet analysis (E). WBCs: white blood cells.