Figure 3.
Figure 3. Identification of a developmental relationship between subpopulations of BM Ly6Chi monocytes. (A) CMap analysis of cMoP, CXCR4hi subset, CXCR4lo subset, and Ly6Clo monocytes displaying their enrichment for Ly6Chi or Ly6Clo monocyte gene sets. (B) OLO reveals a computationally determined developmental order that starts with the cMoP and ends with the CXCR4lo subset as the most mature cell type. (A and B) Data were analyzed using transcriptomic data obtained from three individual mice. (C and D) BM Ly6Chi monocytes subjected to the Wanderlust algorithm using flow cytometry data of six parameters. Data are representative of one out of three independent experiments. (C) Events were plotted against the wanderlust parameter with low and high values representing early and late predicted events, respectively. Early and late events were next plotted for CXCR4 expression against CD11b, CCR2, and CX3CR1. (D) Wanderlust traces showing low CXCR4, CD31, and CD16/32 during the early trajectory (0–0.5), and an increase in CX3CR1, CCR2, and CD11b during the late trajectory (0.5–1). (E) Intrafemoral transfer of sorted CD45.1+ CXCR4hi subset into CD45.2 recipients. Blue dots represent transferred cells after 8 and 24 h. Results are representative of one out of five mice in three independent experiments. (F) FACS plots of BrdU+ (blue) and BrdU− (red) Ly6Chi monocytes after BrdU administration. Results are representative of one out of five independent mice. (G) Alteration in CXCR4 expression over time among BM Ly6Chi monocytes (left) and percentage of BrdU+ Ly6Chi monocytes in the blood (right) at various time points. ΔMFI was calculated by subtracting the CXCR4 MFI of BrdU− Ly6Chi monocytes from that of BrdU+ Ly6Chi monocytes (as described in Fig. S3). Results are expressed as mean ± SEM (n = 5) and representative of one out of two independent experiments. (H) Graph showing correlation between blood BrdU+ Ly6Chi monocytes and ΔMFI of CXCR4 in the BM. Each dot represents one data time point and significance was determined using a Pearson correlation test.

Identification of a developmental relationship between subpopulations of BM Ly6Chi monocytes. (A) CMap analysis of cMoP, CXCR4hi subset, CXCR4lo subset, and Ly6Clo monocytes displaying their enrichment for Ly6Chi or Ly6Clo monocyte gene sets. (B) OLO reveals a computationally determined developmental order that starts with the cMoP and ends with the CXCR4lo subset as the most mature cell type. (A and B) Data were analyzed using transcriptomic data obtained from three individual mice. (C and D) BM Ly6Chi monocytes subjected to the Wanderlust algorithm using flow cytometry data of six parameters. Data are representative of one out of three independent experiments. (C) Events were plotted against the wanderlust parameter with low and high values representing early and late predicted events, respectively. Early and late events were next plotted for CXCR4 expression against CD11b, CCR2, and CX3CR1. (D) Wanderlust traces showing low CXCR4, CD31, and CD16/32 during the early trajectory (0–0.5), and an increase in CX3CR1, CCR2, and CD11b during the late trajectory (0.5–1). (E) Intrafemoral transfer of sorted CD45.1+ CXCR4hi subset into CD45.2 recipients. Blue dots represent transferred cells after 8 and 24 h. Results are representative of one out of five mice in three independent experiments. (F) FACS plots of BrdU+ (blue) and BrdU− (red) Ly6Chi monocytes after BrdU administration. Results are representative of one out of five independent mice. (G) Alteration in CXCR4 expression over time among BM Ly6Chi monocytes (left) and percentage of BrdU+ Ly6Chi monocytes in the blood (right) at various time points. ΔMFI was calculated by subtracting the CXCR4 MFI of BrdU− Ly6Chi monocytes from that of BrdU+ Ly6Chi monocytes (as described in Fig. S3). Results are expressed as mean ± SEM (n = 5) and representative of one out of two independent experiments. (H) Graph showing correlation between blood BrdU+ Ly6Chi monocytes and ΔMFI of CXCR4 in the BM. Each dot represents one data time point and significance was determined using a Pearson correlation test.

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