Inside-out LFA-1 activation is lost in Kindlin-3–deficient LAD-III T cells, whereas partial VLA-4 activation is retained. (A) FACS staining of α_L, β₂, and CXCR4 of LAD-III and control effector T cells. (B) Chemokine signaling in LAD-III lymphocytes. Control and LAD-III effector T cells were left intact or stimulated for 1 to 5 minutes with CXCL12 (10 nM) at 37°C. Cell lysates were immunoblotted with anti–phospho-AKT and anti-talin mAb. (Ci) T cells were stimulated for 5 minutes with PMA (100 ng/mL) or with CXCL12 (10 nM), as in panel B. Expression levels of either the extension-specific epitope KIM127 or the high affinity β₂ headpiece epitope 327C are shown for control and LAD effector T cells before or after stimulation with CXCL12 (c) or with PMA (p). (Cii) 327C epitope expression on control and LAD-III effector T cells suspended either in physiologic medium or in Mg²⁺-EGTA (ethyleneglycoltetraacetic acid) medium (Mg⁺⁺). (D) Basal and CXCL12-stimulated induction of the 327C epitope in LAD-III effector T cells transfected with either GFP or Kindlin-3–GFP. For comparison, CXCL12-stimulated induction of 327C is shown on control effector T cells transfected with GFP. MFI indicates mean fluorescent intensity. (E) Attachment (tethering) and immediate arrest of control and LAD-III effector T cells on ICAM-1-Fc (95 cell adhesion molecule [CAM] sites/μm²) triggered by immobilized CXCL12 (2 μg/mL). The frequency of transient and firm tethers was determined in 2 fields of view, and the depicted values are the mean ± range. The experiment shown is representative of 4. (F) Effects of immobilized CXCL12 or of lymphocyte pretreatment with PMA on LFA-1–dependent adhesion strengthening of control and LAD-III effector T cells. Lymphocytes were settled on ICAM- 1 (95 CAM sites/μm²) for 1 minute and then subjected to incremented shear forces. The percentage of initially settled T cells that resisted detachment from the substrate at the indicated shear forces was determined in 2 fields of view, and values shown represent the mean ± range. The experiment shown is representative of 3. **P < .001 for the compared experimental groups. (G) Attachment and immediate arrest of control and LAD-III effector T cells on VCAM-1 (coated at 1 μg/mL) triggered by immobilized CXCL12 (2 μg/mL) measured at a shear stress of 0.75 dyn/cm² in 2 fields of view. **P < .001 for firm tethers of LAD-III versus control cells. (Right panel) Expression of the activation β₁ epitope HUTS21 on control effector T cells before and after stimulation with CXCL12, PMA, or Mg²⁺-EGTA medium, as described in panel C. (A-F) LAD-III effector lymphocytes were from patient B. (G) LAD-III effectors were from patient A.