CXCR6 Expression Is Important for Retention and Circulation of ILC Precursors

Figure 2

CXCR6-deficient precursors are retained in the bone marrow. (a) Percentages in Lin⁻ (upper panels) and absolute numbers (lower panels) of BM CLP, ILCP, and ILC2P as defined in Figure 1(a) from Rag2^−/−  Cxcr6^+/+ (wt, blue losange), Cxcr6^Gfp/+ (HZ, red square), or Cxcr6^Gfp/Gfp (KO, green triangle) adult mice. (b) Histograms depicting levels of CXCR6-GFP in ILCP (left panel) or ILC2P (right panel) from Rag2^−/−  Cxcr6^+/+ (filled gray), Cxcr6^Gfp/+ (red line), or Cxcr6^Gfp/Gfp (green line) bone marrows. (c) Percentages of CXCR6⁺ cells (upper panels) and absolute numbers of (lower panels) BM ILCP (left panels) and ILC2P (right panels) in Rag2^−/−  Cxcr6^Gfp/+ (HZ, red square) or Cxcr6^Gfp/Gfp (KO, green triangle) adult mice. (d) Cell culture of BM ILCP from Rag2^−/−  Cxcr6^Gfp/+ (upper panels) or Cxcr6^Gfp/Gfp (lower panels) adult mice. Each population is sorted and cultured at 20 cells per well on OP9-DL4 stromal cells with IL-7, c-KitL, and Flt3-L for 7 days and analyzed by flow cytometry. Indicated obtained progenies were analyzed for T-bet, EOMES, RORγt, and GATA-3 expression. (e) Histograms of BM LSK cells (filled gray, selected as Lin⁻ IL-7Rα⁻    Sca-1⁺) and CLP (red line) for Ki67 expression and DAPI levels. (f) Percentages of Ki67⁺ cells (upper panels) and DAPI⁺ cells (lower panels) among BM CLP, ILCP, and ILC2P in Rag2^−/−  Cxcr6^+/+ (wt, blue losange), Cxcr6^Gfp/+ (HZ, red square), or Cxcr6^Gfp/Gfp (KO, green triangle) adult mice. Data are representative of at least 3 experiments ((b), (e): ), or are from one experiment ((d), at least 4 wells of each conditions), or are from 3 pooled experiments ((a), (c), (f), wt: , HZ: , KO: ). In ((a), (c), (f)), each dot represents a single mouse. Statistical data are displayed with mean and SEM (Student’s unpaired bilateral test, ; ; ).