![Figure 6. Motility on CCL21 primes T-cell LFA-1 in both normal and Kindlin-3–deficient T cells, but the primed LFA-1 undergoes TCR-induced activation on ICAM-1 binding only in normal T cells. (A) The fraction of control PB T cells engaged by firm contacts with ICAM-1 (600 sites/μm2)–coated beads during distinct conditions of motility and TCR stimulation. T cells settled on albumin (nonmotile) or allowed to locomote on immobilized CCL21 were exposed to the anti-CD3 mAb (OKT3) or to control mAb. Where indicated, the α/β I allosteric antagonist of LFA-1/ICAM-1 interactions XVA143 (1μM) was included. The number of firm T-cell–bead contacts (> 60 seconds) was divided by the number of total T-cell–bead-encountering events for each experimental condition to yield the fraction of productive contacts. Values are the means ± SD of 4 fields of view. (B) Control (black) compared with Kindlin-3–null (white) PB lymphocytes were allowed to migrate over a CCL21-coated surface toward scattered DCs. (Bi) Each group of T cells was preincubated either in the presence of OKT3 or a control mAb. (Bii) Control (black) or Kindlin-3–null (white) T cells were allowed to migrate on CCL21 toward DCs preloaded with medium or superantigen (staphylococcal enterotoxin A [SEA]). The fractions of T cells that established firm adhesion (> 5 minutes) after colliding into prespread DCs are shown. Values are the means ± SD of 9 fields of view in 3 independent experiments. The frequency of T-cell collision into DCs was comparable in all experimental groups. (C) Control (left) or Kindlin-3–null (right) PB T cells left alone (−, top) or allowed to locomote on immobilized CCL21 (bottom) as in panel A were fixed and stained with either the pan–anti-β2 mAb (TS1.18, left) or the 327C mAb (right) that detects the active open state of the β2 I domain. Merged DIC/fluorescence images of representative T cells are shown. Scale bar represents 3 μm. (D) The fraction of control (black) compared with Kindlin-3–null (white) PB lymphocytes engaged by productive contacts with high-density (4200 sites/μm2) ICAM-1–coated beads under conditions identical to those in panel A. The fraction of T cells suspended in Mg2+/EGTA medium firmly bound to similar ICAM-1 beads is shown for comparison. Values are the means ± SD of 4 fields of view.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/117/26/10.1182_blood-2010-12-322859/5/zh89991173390006.jpeg?Expires=1724230544&Signature=eKg3sf8XRVfz2yHw081RvmQiI-LhQRETjYYTNiO3Iftx8w5J6Qkf3N3dVZxwTfa4YTbGY6~Gs5TrBGXu42vBCnUuqoYObyIxtxwrIJznYywulJd8I1eJrp-sctDuHus8~zF99Oc6yklt12q-Ifl-lHye7gWtqKNcVjrHQqNAMY9c4hcjAO8kTk5wQXOoE7kPSFpoxa6TFGBoUg2nNIKfU3df-WKTUzj7z1OTJFSZydLt6TRin2ke-sNas~38Sy4Bt70JKBePzGrdz1FsHGvv7MiEaofwumxFPvXUYfk9zS7lcqt5t~RhLqskenlFg-68JD2TSPZBri95w53y7RfHmQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Motility on CCL21 primes T-cell LFA-1 in both normal and Kindlin-3–deficient T cells, but the primed LFA-1 undergoes TCR-induced activation on ICAM-1 binding only in normal T cells. (A) The fraction of control PB T cells engaged by firm contacts with ICAM-1 (600 sites/μm2)–coated beads during distinct conditions of motility and TCR stimulation. T cells settled on albumin (nonmotile) or allowed to locomote on immobilized CCL21 were exposed to the anti-CD3 mAb (OKT3) or to control mAb. Where indicated, the α/β I allosteric antagonist of LFA-1/ICAM-1 interactions XVA143 (1μM) was included. The number of firm T-cell–bead contacts (> 60 seconds) was divided by the number of total T-cell–bead-encountering events for each experimental condition to yield the fraction of productive contacts. Values are the means ± SD of 4 fields of view. (B) Control (black) compared with Kindlin-3–null (white) PB lymphocytes were allowed to migrate over a CCL21-coated surface toward scattered DCs. (Bi) Each group of T cells was preincubated either in the presence of OKT3 or a control mAb. (Bii) Control (black) or Kindlin-3–null (white) T cells were allowed to migrate on CCL21 toward DCs preloaded with medium or superantigen (staphylococcal enterotoxin A [SEA]). The fractions of T cells that established firm adhesion (> 5 minutes) after colliding into prespread DCs are shown. Values are the means ± SD of 9 fields of view in 3 independent experiments. The frequency of T-cell collision into DCs was comparable in all experimental groups. (C) Control (left) or Kindlin-3–null (right) PB T cells left alone (−, top) or allowed to locomote on immobilized CCL21 (bottom) as in panel A were fixed and stained with either the pan–anti-β2 mAb (TS1.18, left) or the 327C mAb (right) that detects the active open state of the β2 I domain. Merged DIC/fluorescence images of representative T cells are shown. Scale bar represents 3 μm. (D) The fraction of control (black) compared with Kindlin-3–null (white) PB lymphocytes engaged by productive contacts with high-density (4200 sites/μm2) ICAM-1–coated beads under conditions identical to those in panel A. The fraction of T cells suspended in Mg2+/EGTA medium firmly bound to similar ICAM-1 beads is shown for comparison. Values are the means ± SD of 4 fields of view.
