Figure 2. Marked reduction of CD8α IELs... | Rockefeller University Press

Figure 2.

$Figure 2. Marked reduction of CD8α IELs in GPR18-deficient mixed BM chimeras. (A) B6-CD45.1+ mice were reconstituted with 50% CD45.1/2+ WT and 50% CD45.2+ WT, Gpr18+/−, or Gpr18−/− BM 2–3 mo before analysis. IELs were stained for the subsets as indicated. Numbers indicate percentage of cells in each gate. CD45.1+CD45.2− cells are radiation-resistant host cells. Ratios of WT, Gpr18+/−, or Gpr18−/− CD45.2+ cells versus WT CD45.1/2+ cells were plotted. (B) Ratios of Gpr18−/− versus WT CD8αα γδT IELs in different small intestine segments of mixed chimeras. The ratios were connected for each mouse. (C) Flow cytometry analysis of the indicated markers in the CD8αα γδT IELs from mixed chimeras. Graphs on the right show summary of data from several experiments, where % indicates fraction of cells positive for the marker and MFI indicates mean fluorescence index. Data are representative of more than five experiments in A and three experiments in B and C. (D) Transwell migration assay of CD8αα γδT IELs from mixed chimeras (left and middle) or nonchimeric mice (right) of the type indicated in response to medium alone (nil), 5 µg/ml CCL25, or 1 µg/ml CXCL10, shown as percentage of input cells that migrated. Graph on right shows the fold change in γδT IEL migration in response to CCL25 versus no chemokine. Data are representative of four experiments. (E) Number of CD8αα γδT IELs in WT, Ccr9−/−, and Ccr9−/− Gpr18−/− mice. (F) Frequency of Vγ7+ cells among CD8αα γδT IELs of WT and Ccr9−/− origin in WT:Ccr9−/− mixed chimeras. (G) Intracellular staining of stimulated CD8αα γδT IELs from mixed chimeras for IFN-γ and TNF, shown as frequency of positive cells. (H) Frequency of CD8αα γδT IELs in mixed chimeras that were positive for BrdU staining after 1 wk of in vivo labeling. (I) Frequency of CD8αα γδT IELs in mixed chimeras that were Annexin V+ by flow cytometric analysis. *, P < 0.05; **, P < 0.01; ***, P ≤ 0.001 (Student’s t test). In D, the migration of Gpr18−/− cells was significantly reduced compared with WT (P < 0.001). In A and C–I each symbol represents an individual mouse, and the horizontal lines indicate the mean.$

Marked reduction of CD8α IELs in GPR18-deficient mixed BM chimeras. (A) B6-CD45.1⁺ mice were reconstituted with 50% CD45.1/2⁺ WT and 50% CD45.2⁺ WT, Gpr18^+/−, or Gpr18^−/− BM 2–3 mo before analysis. IELs were stained for the subsets as indicated. Numbers indicate percentage of cells in each gate. CD45.1⁺CD45.2⁻ cells are radiation-resistant host cells. Ratios of WT, Gpr18^+/−, or Gpr18^−/− CD45.2⁺ cells versus WT CD45.1/2⁺ cells were plotted. (B) Ratios of Gpr18^−/− versus WT CD8αα γδT IELs in different small intestine segments of mixed chimeras. The ratios were connected for each mouse. (C) Flow cytometry analysis of the indicated markers in the CD8αα γδT IELs from mixed chimeras. Graphs on the right show summary of data from several experiments, where % indicates fraction of cells positive for the marker and MFI indicates mean fluorescence index. Data are representative of more than five experiments in A and three experiments in B and C. (D) Transwell migration assay of CD8αα γδT IELs from mixed chimeras (left and middle) or nonchimeric mice (right) of the type indicated in response to medium alone (nil), 5 µg/ml CCL25, or 1 µg/ml CXCL10, shown as percentage of input cells that migrated. Graph on right shows the fold change in γδT IEL migration in response to CCL25 versus no chemokine. Data are representative of four experiments. (E) Number of CD8αα γδT IELs in WT, Ccr9^−/−, and Ccr9^−/−Gpr18^−/− mice. (F) Frequency of Vγ7⁺ cells among CD8αα γδT IELs of WT and Ccr9^−/− origin in WT:Ccr9^−/− mixed chimeras. (G) Intracellular staining of stimulated CD8αα γδT IELs from mixed chimeras for IFN-γ and TNF, shown as frequency of positive cells. (H) Frequency of CD8αα γδT IELs in mixed chimeras that were positive for BrdU staining after 1 wk of in vivo labeling. (I) Frequency of CD8αα γδT IELs in mixed chimeras that were Annexin V⁺ by flow cytometric analysis. *, P < 0.05; **, P < 0.01; ***, P ≤ 0.001 (Student’s t test). In D, the migration of Gpr18^−/− cells was significantly reduced compared with WT (P < 0.001). In A and C–I each symbol represents an individual mouse, and the horizontal lines indicate the mean.