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Article
Nature Immunology - 7, 1101 - 1108 (2006)
Published online: 10 September 2006; | doi:10.1038/ni1384

Surface-bound chemokines capture and prime T cells for synapse formation

Rachel S Friedman, Jordan Jacobelli & Matthew F Krummel

Supplementary Fig. 1 (pdf 2M)
CCL21-induced costimulation of naive T cell proliferation is blocked by Pertussis toxin.

Supplementary Fig. 2 (pdf 3M)
Model: Tethering to chemokine-bearing cells allows T cells to pause and survey their environment, and primes T cells for antigen specific coupling and proliferation.

Supplementary Video 1 (mov 2M)
Prototypical antigen dependent coupling. Representative image of T cell immunological synapse on an antigen-bearing APC as quantified in Fig. 3b. Time-lapse series of DIC (left) and Fura-2AM calcium (right) images of labeled T cell blasts interacting with A20 B cell APCs in the presence of 1mug/ml OVA peptide. The T cell contacts the APC with its leading edge, fluxes calcium and shows the canonical IS morphology – rounded with a flat interface. Calcium flux is on a pseudocolor scale indicating the ratio of Fura-2AM emissions at 340nm/380nm. A timestamp (bottom) indicates the elapsed time in minutes:seconds.

Supplementary Video 2 (mov 1M)
CCL21 induced T cell tethering. Movie version of Fig. 2a. Representative image of T cell tether on a chemokine-bearing APC as quantified in Fig. 2b. A timestamp (bottom) indicates the elapsed time. DIC (left) and corresponding FURA-2AM ratio (right) are shown.

Supplementary Video 3 (mov 2M)
CCL21 induced two-step T cell activation with a tethered intermediate on an antigen-bearing APC. Movie version of Figure 3a. Representative image of T cell tethering followed by immunological synapse formation on an antigen and chemokine-bearing APC as quantified in Fig. 3b. A timestamp (bottom) indicates the elapsed time. DIC (left) and corresponding FURA-2AM ratio (right) are shown.

Supplementary Video 4 (mov 2M)
Two-step T cell activation induced by endogenously produced chemokine on antigen bearing DCs. Timelapse series of DIC (left) and Fura-2AM calcium (right) images of labeled T cell blasts interacting with bone-marrow derived DCs in the presence of 1mug/ml OVA peptide. The T cell contacts the APC with its leading edge, crawls along the APC, and remains attached or tethered to the APC via its uropod. The tethered T cell maintains its polarized amoeboid morphology with an active leading edge and constricted uropod and does not flux calcium until a second contact with a DC induces calcium flux and immunological synapse formation. The cells two of interest are indicated by white and yellow the arrows. Calcium flux is on a pseudocolor scale indicating the ratio of Fura-2AM emissions at 340nm/380nm. A timestamp (top) indicates the elapsed time in minutes:seconds.

Supplementary Video 5 (mov 2M)
CCL21 induced tethering in naïve T cells. Representative image of T cell tether on a chemokine-bearing APC as quantified in Fig. 6a. Timelapse series of DIC (left) and Fura-2AM calcium (right) images of labeled naïve DO11.10 T cell interacting with bone-marrow derived DCs in the presence of 1mug/ml CCL21. The T cell contact the APC and remain bound to it. While bound, the T cells fluctuate between a rounded morphology and a polarized morphology characteristic of a tethered T cell blast. Calcium flux is on a pseudocolor scale indicating the ratio of Fura-2AM emissions at 340nm/380nm. A timestamp (top) indicates the elapsed time in minutes:seconds.


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Nature Immunology
ISSN: 1529-2908
EISSN: 1529-2916
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