Figure 7. FAK1 reduces T-cell contact times with DCs, an effect dependent on the LAT Y-171.

(a) Dot plots showing that LAT-Y-171F expression reverses FAK1 inhibition of T-cell dwell times. DO11.10 T cells cells were prepared as above and co-transfected with FAK1 and LAT or LAT-Y171F. Mature DCs were labelled with SNARF-1 and pre-incubated with OVA peptide (DC-OVA) prior to incubation with T cells. (b) Dot plots showing that LAT-Y-171F expression reverses FAK1 increase in T-cell velocity. DO11.10 T cells were prepared as above. (c) Dot plots showing that LAT-Y-171F expression reverses FAK1 increase in T-cell displacement. DO11.10 T cells were prepared as above. (d) LAT-Y-171F expression alone increases T-cell dwell times. LAT-Y171F increases T-cell contact (left panel); LAT-Y171F decreases T-cell motility (middle panel); LAT-Y171F decreases T-cell displacement (middle panel). D011.10 T cells were prepared as above and co-transfected with LAT-Y171F prior to a measure of T-cell contact with DCs. (e) LAT-Y171F expression reverses FAK1 inhibition of DO11.10T-cell proliferation in response to OVA peptide. DO11.10 T cells were prepared as above except that they were pre-labelled with CFSE. Left panels: FACs histograms of CFSE labelling; right histogram: panels showing the inhibition of T-cell cycling by FAK1 LAT-WT (gold columns showing most cells in M1–M3, whereas FAK1 LAT Y-171F shows the increased presence of cells in M3-M6 (n=4). (f) LAT-Y-171F expression blocks T-cell motility/migration on ICAM1-Fc coated plates. J14 Jurkat cells were transfected with LAT or LAT-Y-171F and tracked for migration. Left panel: immunoblots of lysates transfected with LAT or LAT-Y-171F; middle panel: tracking profiles of LAT transfected J14 T cells; right panel: tracking profiles of LAT-Y-171F transfected J14 T cells (n=3). P values were calculated by one-way analysis of variance (ANOVA) between groups followed by a series of t-tests.