Cytometry by time of flight identifies 2 neutrophil clusters that are increased in mice that received CpG-MSCs compared with mice that received PBS; flow cytometry further identifies a CD150⁻CD48⁺ restricted progenitor population that is expanded in mice that received CpG-MSC. Single cell suspensions from the bone marrow of irradiated mice that received PBS (n = 4), MSC (n = 5), and CpG-MSC (n = 5) prepared 48 hours after infection with P aeruginosa were labeled with 33 immunophenotyping markers. (A) A tSNE plot of concatenated data from PBS, MSC, and CpG-MSC groups after equal sampling (10 000 events per file). Manual gating identified distinct clusters of Ly6G⁺ neutrophils (orange), TER-119⁺ erythroid cells (green), CD19⁺ B cells (purple), CD3⁺ T cells (red), NK cells (pink), and F4/80⁺ macrophages (brown). Cells not otherwise identified by immunophenotyping markers are represented in blue. (B) Forty-five clusters were identified using SPADE algorithm and overlaid on a tSNE plot of concatenated data. (C) Data were assessed by 2-way ANOVA, interaction P = .0002. 2 clusters, cluster 28 (red) and cluster 23 (green) were further identified as significantly different between PBS and CpG-MSC groups, but not PBS vs MSC, by post hoc multiple comparisons testing. (D) Event counts of the number of cells in cluster 23 (○, pink bars) and cluster 28 (▪, blue bars) in mice that received PBS, MSC, and CpG-MSC are shown. Post hoc analysis by Dunnett’s multiple comparison test was performed. *Significant comparisons, PBS vs CpG-MSC, cluster 23, P = .029); **PBS vs CpG-MSC, cluster 28, P = .003. (E) Log-transformed mean expression levels of select immunophenotyping markers (columns) by cluster number (rows) in clusters 23 and 28 from data concatenated from irradiated and infected mice given PBS (n = 4), MSC (n = 5), and CpG-MSC (n = 5). Flow cytometric analysis of primitive progenitors demonstrate no significant difference between mice given PBS (n = 8, green filled circle), MSC (n = 9, blue filled square), and CpG-MSC (n = 9, red filled triangle) by ANOVA in the numbers of (F) long-term hematopoietic stem cells (LT-HSC; Lin⁻Sca1⁺C-kit⁺CD150⁺CD48⁻) or (H) multipotent progenitors (MPP; Lin⁻Sca1⁺C-kit⁺CD150⁻CD48⁻) cells. (I) There was an increased number of Lin⁻Sca1⁺C-kit⁺CD150⁻CD48⁺ restricted progenitor cells (HPC-1) but not (G) Lin⁻Sca1⁺C-kit⁺CD150⁺CD48⁺ restricted progenitor cells (HPC-2) in mice that received CpG-MSC. Data were analyzed by ANOVA, P = .002. *Significant comparisons by Bonferroni’s multiple comparisons test, PBS vs CpG-MSC, P = .005; §CpG-MSC vs MSC, P = .010. tSNE, t-distributed stochastic neighbor embedding.