Reduction of CD8αα γδT IELs in GPR18-deficient mice. (A) QPCR analysis of Gpr18 mRNA transcript abundance in spleen, liver, and sorted spleen cells and small intestine (SI) IELs as indicated, presented relative to Hprt. (B) Total SI IEL numbers of Gpr18^+/− and Gpr18^−/− mice. (C) Percentage of indicated subsets among total IELs from Gpr18^+/− and Gpr18^−/− mice. CD8αα cells were gated as CD8α⁺CD8β⁻. Mice in B and C were 2–3 mo old. ***, P ≤ 0.001 (Student’s t test). (D) Total SI IEL numbers in aged (∼7 mo old) Gpr18^+/− and Gpr18^−/− mice. *, P < 0.05 (Student’s t test). (E) Transwell migration assay of WEHI231 cells transduced with the retroviral vectors as labeled, in response to the indicated ligands. CXCL12 concentration was 100 ng/ml. (A and E) Data are shown as mean ± SD. EV, empty vector; NAGly, N-arachidonoyl-glycine; 2-AG, 2-arachidonyl-glycerol. (F) Immunofluorescent staining of IELs in duodenum from Gpr18^+/− and Gpr18^−/− mice. Arrowheads highlight CD8α and γδTCR double-positive (yellow) cells. (G) Flow cytometric analysis of IELs and LPLs from a mouse injected 5 h earlier with anti–CD8α-PE. (H) Time-lapse images of duodenum of Gpr18^+/− and Gpr18^−/− mice. CD8α-PE (red) and nuclei (DAPI, blue) are shown. Dashed line indicates the interface between the epithelium and the intestinal lumen. Arrowheads point to the location of one tracked CD8α⁺ cell at each time point, and lines indicate tracked paths. Bars: (F) 100 µm; (H) 10 µm. In B–D each symbol represents an individual mouse, and the horizontal line indicates the mean. Data in A, E, F, and H are representative of three experiments.