Fig. 4.

TCR-induced LFA-1 dependent T cell adhesion depends on the SLAT PH domain. (A,B) Jurkat JA16T cells were transfected with empty pEF vector or pEF-Myc-SLAT (10 µg each) plus the indicated amounts of Myc-tagged SLAT-ΔPH mutant (A,B) or SLAT-R236C mutant (B). Cells were either left unstimulated (NS) or stimulated with OKT3 mAb (TCR) for 45 min, and subsequently analyzed for adhesion to plate-bound Fc-ICAM-1. Adhesion data represents the mean±s.d. of four independent experiments. Lower panels, expression of transfected proteins was detected by anti-Myc immunoblotting. An anti-β-actin immunoblot serves as a loading control. (C–E) Primary WT and Def6^−/− (KO) CD4⁺ T cells were activated with anti-CD3 plus anti-CD28 mAbs and IL-2 and transduced with retroviral pMIG vectors expressing either GFP alone (Empty) or GFP plus the indicated SLAT cDNAs. Sorted GFP⁺ CD4⁺ T cells were left unstimulated (NS) or restimulated with anti-CD3 mAb (TCR) for 3 min (C,D) or 45 min (E), and analyzed either for their ability to bind soluble Fc-ICAM-1 by flow cytometry (C,D) or for their ability to adhere to plate-bound Fc-ICAM-1 (E). Numbers shown in FACS histograms (C) indicate the percentage of GFP⁺ CD4⁺ T cells able to bind soluble Fc-ICAM in a representative experiment. Data shown are representative of four independent experiments. (D,E) ICAM-1 binding and adhesion data represent the mean±s.d. of the percentage of four independent experiments. *P<0.01, WT versus Def6^−/− cells; ns, not significant (two-tailed Student's t-test).