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Blood, Vol. 113, Issue 24, 6138-6147, June 11, 2009
The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration
Blood Gérard et al.
113: 6138
Supplemental materials for: Gerard et al
Files in this Data Supplement:
- Figure S1. Identification of transmigrating T cells through an endothelial monolayer (JPG, 109 KB)
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Criteria to determine lymphocyte transmigration were based on analysis of the relative distribution of a set of markers, including actin, ICAM-1, LFA-1, ZO-1, PECAM-1, and VE-Cadherin in both the x-y and the z dimensions. (A) T cells crawling on the top of the endothelial monolayer were visualized on the same plane or above tight junctions (TJ) (visualized by ZO-1), in a more apical site relative to adherent junctions (visualized by VE-Cadherin). A transmigrating cell, however, would be detected all the way down to the basal side of the endothelial monolayer. Alternatively, the position of lymphocytes in relation to the endothelial cell could be easily judged by determining whether stress fibers in the endothelial cell were located above or below the T cell as reported by Sandig et al (J Cell Sci 1997 110: 2807–2818). (B) Example of the identification of a crawling and transmigrating T cell. Cells were stained with ZO-1 Ab and phalloidin to visualize TJs and stress fibers. T cells are visualized with LFA-1 staining and phalloidin. The crawling cell is found on the apical site of the endothelial monolayer, above stress fibers, whereas a cell in the process of transmigrating appears above as well as underneath the TJs, down to the most basal site, with protrusion present underneath the endothelial stress fibers.
- Figure S2. Tiam1 is required for homing of CD4- and CD8-positive T cells to SLOs (JPG, 45.4 KB)
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CFDA-labeled WT and CMTMR-labeled Tiam1−∕− naïve T cells were mixed at a ratio 1:1 and 20 millions of cells were injected intravenously in WT mice. Two hours after transfer, cells obtained from pLNs, mesenteric LNs, spleen and blood were stained for CD4 or CD8 surface marker, and the percentage of Tiam1−∕− and WT labeled T cells present in every organ was calculated by FACS analysis. Results are expressed as a homing ratio, corresponding to the ratio between the number of Tiam1−∕− and WT cells. Bars are means. Every single experiment is depicted by a dot. No significant difference was observed between the CD4 and CD8 population.
- Figure S3. Tiam1−∕− T cells have a destabilized leading edge, resulting in decrease directionality and speed (JPG, 96.7 KB)
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(A) WT and Tiam1−∕− T-cell blasts were retrovirally transduced with a vector encoding GFP-Actin. Cells were subjected to shear flow on ICAM-1 substrate during 10 minutes. Images were recorded every 20 seconds. Representative GFP-Actin localization during migration of adherent WT and Tiam1−∕− T-cell blasts are shown. Scale bar: 5μm. Arrows indicate the direction of migration. (B) WT and Tiam1−∕− T-cell blasts were subjected to shear flow on ICAM-1 substrate and migration was tracked over a 8-min period, where one picture was taken every 6 seconds. Tracking of cells on a 3 minutes period (average period where a cell is present on the acquired field) was manually done using the Image J software. X and Y coordinates correspond to the location of a cell compared to its starting point, and is expressed in mm. Each line represents one cell. (C) WT and Tiam1−∕− T-cell blasts were subjected to shear flow on ICAM-1 substrate and migration was tracked over a 10-min period, where one picture was taken every 6 seconds. Speed is expressed in mm/min and was deduced using the Image J software. Error bars indicate SD; p means p-value.
- Figure S4. Tiam1 is required for crawling of naive T cells (JPG, 37.6 KB)
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After an accumulation phase on activated bEnd.3 monolayer (2 minutes at 0.2 dyne/cm2), naive WT- and Tiam1−∕− T cells were subjected to shear flow (2 dynes/cm2) for 15 minutes. Three independent experiments were performed and at least two fields per condition for each experiment were recorded. The behaviour of single adherent T cell was analyzed and classified as stationary, crawling (without trans-endothelial migration), or transmigrating (diapedesis condition). Values indicate mean +∕− SD; p indicates p-value between the percentage of WT and Tiam1−∕− T cells.
- Figure S5. The average time required for a single WT- and Tiam1−∕− T cell to cross the endothelial monolayer is identical (JPG, 24.1 KB)
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After an accumulation phase on activated bEnd.3 monolayer (2 minutes at 0.2 dyne/cm2), T-cell blasts were subjected to shear flow (2 dynes/cm2) for 15 minutes. Two independent experiments were performed and three fields per condition for each experiment were recorded, in which more than 30 cells were analyzed. Average time required for a single T cell to cross the endothelial monolayer was manually calculated. Values indicate mean +∕− SD; p indicates p-value between WT and Tiam1−∕− T cells. No significant difference in time was observed between WT and Tiam1−∕− T cells.
- Video 1. Time-lapse video microscopy of arrest and migration of WT T-cell blasts on ICAM-1 surface in the presence of Ca2+/Mg2+ (AVI, 4.09 MB)
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Video is set to 8 frames/s each with a frame rate of 6s.
- Video 2. Time-lapse video microscopy of arrest and migration of Tiam1−∕− T-cell blasts on ICAM-1 surface in the presence of Ca2+/Mg2+ (AVI, 7.74 MB)
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Video is set to 8 frames/s each with a frame rate of 6s.
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