Figure 4. Rap1 deficiency enhances rolling of T_EM cells.

(a) Representative expression profiles of CD44 and CD62L, α₄β₇, LFA-1, PSGL-1 and CXCR3 in CD4⁺T cells from f/f or −/− mice, cultured under T_H17-polarizing conditions. (b) f/f T_EM cells were incubated at 37 °C at 15 s in the presence or absence of CXCL10. Cell lysates were pulled down with GST-RalGDS-RBD and immunoblotted with anti-Rap1. (c; Left) f/f or −/− T_EM cells were perfused on LS12 cells expressing P-selectin and mouse ICAM-1 ±CXCL10. Adhesion of more than 100 cells was measured. *¹P<0.002, *²P<0.05 versus total frequency of the corresponding f/f T_EM cells. (Right) The rolling velocity of f/f or −/− T_EM cells without CXCL10. *P<0.03 versus f/f T_EM cells. (d; Left) f/f or −/− T_EM cells were perfused on LS12 cells expressing mouse MadCAM-1 ±CXCL10. Adhesion of more than 100 cells was measured. *¹P<0.001, *²P<0.002 versus total frequency of the corresponding f/f T_EM cells. (Right) The rolling velocity of f/f or −/− T_EM cells without CXCL10. *P<0.01 versus f/f T_EM cells. (e; Left) f/f or −/− T_EM cells were perfused on plates immobilized with MadCAM-1 ±CXCL10. Adhesion of more than 100 cells was measured. *¹P<0.02 versus unstimulated f/f T_EM cells. *²P<0.01, *³P<0.03 versus total frequency of the corresponding f/f T_EM cells. (Right) The rolling velocity of f/f or −/− T_EM cells on MadCAM-1 without CXCL10. *P<0.005 versus f/f T_EM cells. (f) Homing assay of T_EM cells into the colon. (Left) f/f or −/− T_EM cells labelled with CMTMR or CFSE were injected into normal mice. After 10 h, cells from the colon LP of recipient mice were analysed using flow cytometry. The ratios of −/− to f/f T_EM cells are shown (n=3 experiments). (Right) Flow-cytometry profile of colon LP in the recipient mice. Numbers indicate the ratio of −/− to f/f T_EM cells. All data show the means±s.e.m. or are representative of three independent experiments.