Dendritic Cell Stimulation by Mycobacterial Hsp70 Is Mediated Through CCR5
R. Andres Floto, Paul A. MacAry, Jessica M. Boname, Tan Suet Mien, Beate Kampmann, James R. Hair, Oh Seen Huey, Edith N. G. Houben, Jean Pieters, Cheryl Day, Wulf Oehlmann, Mahavir Singh, Kenneth G. C. Smith, Paul J. Lehner

Supporting Online Material

This supplement contains:
Materials and Methods
Figs. S1 to S7
Table S1
Videos S1 to S4
References

Correction (31 October 2006): Table S1 and the graphic images for Figs. S1 to S7 were missing from the original version of the SOM and have been added to the file.

This file is in Adobe Acrobat PDF format.

Video S1.
The effect of myHps70 on iDC calcium signaling and aggregation. Merged simultaneous fluorescence and DIC images (taken every 20s ) of Fluo3-labelled iDC placed on uncoated coverslips during addition of myHsp70 (200 nM). Addition of myHsp70 induces rapid calcium signalling immediately followed by membrane ruffling and extensions and cellular aggregation. Video at 2 frames/s.

Video S2.
The effect of myHsp70 on aggregation of iDC with autologous CD4+ T cells. Merged simultaneous fluorescence and DIC images (taken every 20 s) of Fluo3-labelled iDC placed on poly-L-lysine coated coverslips with autologous CD4+ T cell (labeled with 5 mM calcium orange-AM) during addition of myHsp70 (200 nM). Addition of myHsp70 results in DC calcium signalling, rapid membrane ruffling and extension of pseudopodia which trap passing T cells leading to stable DC-T cell clusters. Video at 2 frames/s.

Video S3.
The effect of myHsp70 on aggregation of iDC with autologous CD8+ T cells. Merged simultaneous fluorescence and DIC images (taken every 20 s) of Fluo3-labelled iDC placed on poly-L-lysine coated coverslips with autologous CD8+ T cell (labelled with 5 mM calcium orange-AM) during addition of myHsp70 (200 nM). Video at 2 frames/s.

Video S4.
The effect of myHsp70 on aggregation of iDC and CD8+ T cells in the absence of surface CCR5 expression. Merged simultaneous fluorescence and DIC images (taken every 20 s) of Fluo3-labelled CCR532 iDC placed on poly-L-lysine coated coverslips with autologous CCR532 CD8+ T cell (labelled with 5 mM calcium orange-AM) during addition of myHsp70 (200 nM). Addition of myHsp70 causes neither a calcium response nor membrane extensions from DC to T cells. Video at 2 frames/s.

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