American Society of Hematology

Figure 4

$Figure 4. TCR-triggered Kindlin-3–null T cells fail to spread on DCs. (A) ICAM-1 expression on human DCs spread for 1 hour on immobilized CCL21 before being tested as a substrate for PB T cells. DCs were fixed and stained with PE-conjugated α-ICAM-1 mAb. Staining of resting and TNFα-stimulated HUVECs (200 U/mL for 24 hours) is shown for comparison. Scale bar represents 10 μm. (Bi) PB T cells were stimulated with OKT3 (10 μg/mL) or left in medium (–) and settled alone or in the presence of the indicated inhibitors on DCs prespread on immobilized CCL21 as in panel A. For each experimental group, the fractions of settled lymphocytes that spread on the DCs were quantified by live videomicroscopy, as described in “Methods.” Values are the means ± SD of 9 fields of view in 3 independent experiments. ***P < 10−6. (Bii) CD4+ PB T cells were settled for 10 minutes on DCs that had been left untreated or preloaded for 1 hour with staphylococcal endotoxin A (1 μg/mL). All DCs were prespread on immobilized CCL21 as in panel A. The fractions of lymphocytes spread on DCs were quantified as in panel Bi. Values are the means ± SD of 4 fields of view in 1 experiment representative of 3. **P < .005. (Ci) Normal PB T cells were incubated with OKT3 (10 μg/mL), labeled with a trace of Alexa Fluor 568–labeled TS2.4 (anti–αL, 1 μg/mL), and allowed to spread for 5 minutes on DCs before fixation. Shown are a DIC image (left) and a fluorescence image (right) of LFA-1 in a representative T cell. Scale bar represents 3 μm. (Cii) Fluorescence staining of LFA-1 visualized by Alexa Fluor 568–labeled TS2.4 anti–LFA-1 in a normal CD4+ T cell spread for 10 minutes on an staphylococcal endotoxin A–loaded DC prespread on CCL21 and fixed. Scale bar represents 3 μm. (D) Transmission electron microscopic image of a representative OKT3-stimulated normal T cell spread on a DC prespread on immobilized CCL21 as in panel Bi. Because no Kindlin-3–null T cells could be recovered from the DC after fixation, similar fluorescence staining and electron microscopy could not be carried out for these T cells. Scale bar represents 3 μm.$

TCR-triggered Kindlin-3–null T cells fail to spread on DCs. (A) ICAM-1 expression on human DCs spread for 1 hour on immobilized CCL21 before being tested as a substrate for PB T cells. DCs were fixed and stained with PE-conjugated α-ICAM-1 mAb. Staining of resting and TNFα-stimulated HUVECs (200 U/mL for 24 hours) is shown for comparison. Scale bar represents 10 μm. (Bi) PB T cells were stimulated with OKT3 (10 μg/mL) or left in medium (–) and settled alone or in the presence of the indicated inhibitors on DCs prespread on immobilized CCL21 as in panel A. For each experimental group, the fractions of settled lymphocytes that spread on the DCs were quantified by live videomicroscopy, as described in “Methods.” Values are the means ± SD of 9 fields of view in 3 independent experiments. ***P < 10⁻⁶. (Bii) CD4⁺ PB T cells were settled for 10 minutes on DCs that had been left untreated or preloaded for 1 hour with staphylococcal endotoxin A (1 μg/mL). All DCs were prespread on immobilized CCL21 as in panel A. The fractions of lymphocytes spread on DCs were quantified as in panel Bi. Values are the means ± SD of 4 fields of view in 1 experiment representative of 3. **P < .005. (Ci) Normal PB T cells were incubated with OKT3 (10 μg/mL), labeled with a trace of Alexa Fluor 568–labeled TS2.4 (anti–α_L, 1 μg/mL), and allowed to spread for 5 minutes on DCs before fixation. Shown are a DIC image (left) and a fluorescence image (right) of LFA-1 in a representative T cell. Scale bar represents 3 μm. (Cii) Fluorescence staining of LFA-1 visualized by Alexa Fluor 568–labeled TS2.4 anti–LFA-1 in a normal CD4⁺ T cell spread for 10 minutes on an staphylococcal endotoxin A–loaded DC prespread on CCL21 and fixed. Scale bar represents 3 μm. (D) Transmission electron microscopic image of a representative OKT3-stimulated normal T cell spread on a DC prespread on immobilized CCL21 as in panel Bi. Because no Kindlin-3–null T cells could be recovered from the DC after fixation, similar fluorescence staining and electron microscopy could not be carried out for these T cells. Scale bar represents 3 μm.

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