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Blood, Vol. 109, Issue 1, 168-175, January 1, 2007
The actin cloud induced by LFA-1–mediated outside-in signals lowers the threshold for T-cell activation
Blood Suzuki et al.
109: 168
Supplemental materials for: Suzuki et al, Vol 109, Issue 1, 168-175
Files in this Data Supplement:
- Figure S1. Actin cloud formation upon stimulation with stimulatory anti–LFA-1 (PDF, 69.6 KB) -
Jurkat cells were stimulated with 1 µg/mL plate-coated anti–LFA-1 mAb (clone R7.1) or nonstimulatory anti–LFA-1 mAb (clone T1/18) for 30 minutes. Fixed cells were stained with phalloidin–Alexa 568. White arrow indicates actin cloud.
- Figure S2. Actin cloud formation at the interface of T cells and B cells (PDF, 172 KB) -
Jurkat cells (left panels) and IL-2–cultured human PBLs (right) were stimulated with SDF-1 and conjugated with Raji B cells for 30 minutes in the absence of Ag. Stimulated cells were stained with phalloidin–Alexa 488 and anti-CD19–FITC. Images in both x-y (top) and x-z (bottom) axes are shown. Surface staining of CD19 showed that most of the accumulated actin in the interface was derived from T cells, and actin accumulation was not observed in B cells under this condition.
- Figure S3. Actin cloud formation may be required for mature IS formation (PDF, 50.9 KB) -
(A) JNK inhibitor blocks mature IS formation. Jurkat cells were pretreated with three MAPK inhibitors (left margins) similar to Figure 5 and conjugated with SEE-pulsed Raji cells. T-cell–B-cell conjugates were fixed and stained with Abs for CD3, LFA-1, or actin, as indicated. The staining data of Jurkat cells treated with JNK inhibitor (JNK) or untreated (None) were shown. (B) Quantification of panel A. The percentage of Jurkat cells bearing each molecule recruited within IS among cells showing conjugate formation with APCs (right panels) was calculated and shown. More than 200 cells each were analyzed.
- Figure S4. Real-time imaging of actin cloud formation upon anti–LFA-1 stimulation (PDF, 76.9 KB) -
Jurkat cells expressing actin-GFP were plated dropwise on a glass dish coated with anti-CD3 or anti–LFA-1. Images were collected after the actin ring was just formed (several minutes after contact with plate) for 10 minutes using confocal microscopy. White arrows indicate representative actin clouds.
- Figure S5. Transient appearance of the actin cloud during the early course of Ag-induced IS formation (PDF, 87.4 KB) -
Jurkat cells were transiently transfected with actin-GFP and added dropwise to Raji pulsed with SEE. Dynamic movement of actin was monitored in real time by confocal laser microscopy. White arrow indicates actin cloud formation, particularly at 30 seconds.
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