β2TTT/AAA integrin knock-in T cells display impaired adhesion. (A) Adhesion of WT and KI-naïve CD4 T cells to ICAM-1 under static conditions. Cells were unstimulated, or stimulated with 200 nM PDBu or 10 μg/mL anti-CD3 (N = 4). (B) SDF-1–induced adhesion of naïve CD4 T cells to ICAM-1 under shear flow conditions (N = 4). (C) The expression of αL (left) and β2 (right) integrin subunits in WT (solid line) and KI (dashed line)-naïve CD4 T cells ex vivo (top) and cultured effector CD4 T cells (bottom). Plots are representative of N = 3 to 5. Shaded line represents the isotype control. (D) Kindlin-3, β2 integrin and 14-3-3 protein levels in WT and KI effector CD4 T cells, with and without stimulation with 200 nM PdBu. N = 1 is shown as representative of N = 2. (E) β2 integrin mRNA levels in WT and KI-naïve and effector CD4 T cells were determined by reverse-transciptase quantitative PCR (N = 4). (F) Effector CD4 T-cell adhesion to ICAM-1 under static conditions, using unstimulated, PDBu– or anti-CD3–stimulated cells (N = 5). (G) Effector CD4 T-cell adhesion to ICAM-1 under shear flow conditions (N = 4). (H) Effector CD4 T-cell adhesion to bEnd.3 cells under shear flow, after 10 minutes of adhesion (N = 3). (I) Rolling rates of effector CD4 T cells on ICAM-1 under shear flow (N = 3). (J) p-PLCγ and Src levels in effector CD4 T cells were determined by Western blot analysis. N = 2 is shown and is representative of N = 4. Student t test was used to calculate significance values in panels A,E-F,H-I. Two-way analysis of variance was performed in panels B,G. NS, not significant. In all cases, mean ± SEM is shown.
