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Blood, Vol. 107, Issue 8, 3212-3220, April 15, 2006
Long-term engagement of CD6 and ALCAM is essential for T-cell proliferation induced by dendritic cells
Blood Zimmerman et al.
107: 3212
Supplemental materials for: Zimmerman et al
Files in this Data Supplement:
- Figure S1. ALCAM is expressed on CD11c+ peripheral blood DCs (JPG, 39.4 KB)
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Blood DCs were isolated from PBMC by using the MACS Blood DC isolation kit (CLB, Amsterdam, The Netherlands). CD11c+ blood DCs were matured by culturing in 50% monocyte-conditioned medium for 3 days. ALCAM expression was analyzed by flowcytometry. The bar indicates positive staining. One representative experiment out of three is shown.
- Figure S2. Blocking antibodies do not induce toxicity in DC-T cell co-cultures (JPG, 53 KB)
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105 PBL were stimulated in vitro with 1.7 × 103 mature monocyte-derived DCs, in the presence or absence of antibodies against LFA-1 (NKI-L15, 10 µg/ml), CD6 (5 µg/ml), or total mouse IgG (mIgG, 10 µg/ml). Antibodies were added at co-culture onset and propidium iodide (PI) staining was performed at day 5. Cells were analyzed by flowcytometry and gates were set to include PBLs. Clear histograms represent cells without PI, shaded histograms represent PI staining. Percentages of positive cells and MFI values are indicated in the upper right corner of each panel. One representative experiment out of three is shown.
- Video 1A. Dynamic redistribution of ALCAM and CD6 during the formation of T cell-APC conjugates (MOV, 2.84 MB)
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Jurkat-CD6*RFP cells were allowed to adhere for 10 min on fibronectin-coated glass slides. K562-ALCAM*GFP cells were added in a 1:2 ratio and cells were analyzed for 60 min at 37°C with a Zeiss LSM 510 microscope. Images were taken at 20 s intervals and processed with Image J version 1.32j software. Movies show two examples (A and B) of Jurkat-CD6*RFP/K562-ALCAM*GFP contacts.
- Video 1B. Dynamic redistribution of ALCAM and CD6 during the formation of T cell-APC conjugates (MOV, 2.37 MB)
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Jurkat-CD6*RFP cells were allowed to adhere for 10 min on fibronectin-coated glass slides. K562-ALCAM*GFP cells were added in a 1:2 ratio and cells were analyzed for 60 min at 37°C with a Zeiss LSM 510 microscope. Images were taken at 20 s intervals and processed with Image J version 1.32j software. Movies show two examples (A and B) of Jurkat-CD6*RFP/K562-ALCAM*GFP contacts.
- Video 2. Dynamic redistribution of ALCAM during contact formation between two K562-ALCAM*GFP cells (MOV, 2.95 MB)
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Cells were analyzed for 60 min at 37°C with a Zeiss LSM 510 microscope. Images were taken at 20 s intervals and processed with Image J version 1.32j software.
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