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PNAS 105 (22): 7791-7796

Copyright © 2008 by the National Academy of Sciences.

Micropatterning of costimulatory ligands enhances CD4+ T cell function

Keyue Shen*, V. Kaye Thomas{dagger}, Michael L. Dustin{dagger}, and Lance C. Kam*,{ddagger}

*Department of Biomedical Engineering, Columbia University, New York, NY 10027; and {dagger}Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016


Figure 1
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  		Fig. 1. T cell interaction with micropatterned, costimulatory arrays. (A) CD4+ cells were presented with surfaces that capture the microscale organization of ligands associated with T cell costimulation. Colocalized patterns were created by mixing anti-CD3 and anti-CD28 antibodies (yellow) in a single step (B), while segregated patterns were defined by sequential patterning of anti-CD3 (red) and anti-CD28 (green) on a single surface (C). (Inset) Fluorescence profile across a segregated site. ICAM-1 was coated onto the remainder of these surfaces but is omitted here for clarity. (Scale bar: 10 µm.)

 

Figure 2
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  		Fig. 2. Interaction of CD4+ T cells with micropatterned arrays. (A) In this series of images, features of anti-CD3 (larger, 2-µm dots) and anti-CD28 (smaller, 1-µm dots) are shown in red. Cells were seeded onto surfaces 30 min before collection of this time series. A full movie is shown as Movie S1. (Scale bar: 10 µm.) (B) Localization of TCR (detected by using an H57 antibody) and CD28 (antibody against the cytosolic domain) follows the patterned antibodies. (Scale bar: 5 µm.) (C) Cells on segregated surfaces interacted predominantly with three or four anti-CD28 features (see text).

 

Figure 3
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  		Fig. 3. Modulation of IL-2 secretion. (A) IL-2 secretion (green) over 6 h was measured on a cell-by-cell basis. Patterns of anti-CD3 and anti-CD28 antibodies are shown in red. (Scale bar: 25 µm.) (B) Histogram of IL-2 secretion from a representative experiment. IL-2 secretion on each of these conditions is statistically different from the other two (both ANOVA and Kruskal–Wallis analysis, {alpha} = 0.01).

 

Figure 4
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  		Fig. 4. Modulation of NF-{kappa}B translocation. (A) NF-{kappa}B within the core of the nucleus was measured by segmentation of an image stack. (B) Box plots of average NF-{kappa}B in the cell nucleus. The whiskers and elements of the boxes correspond to 5, 25, 50, 75, and 95 percentiles of the data, whereas the diamond corresponds to the dataset average. Each condition is statistically different from the other two ({alpha} = 0.01).

 

Figure 5
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  		Fig. 5. PKC{theta} localization is similar across patterns. Patterned antibodies are shown in red, and PKC{theta} is shown in green. Cell outlines at the substrate interface were generated from long-exposure images and are indicated by dotted lines. (Scale bar: 7 µm.)

 

Figure 6
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  		Fig. 6. Inhibition of Akt. (A) Six-hour secretion of IL-2 in the presence of the Akt-inhibitor triciribene. No significant difference was detected between experiments indicated by asterisks ({alpha} = 0.05). All other comparisons indicated significant differences ({alpha} = 0.01). (B) PI3K (green, showing p85{alpha},β subunits) distribution on colocalized and segregated patterns. Patterns of anti-CD3 and anti-CD28 ligands are shown in red. Cells were fixed 10 min after seeding. (Scale bar: 5 µm.)

 

Figure 7
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  		Fig. 7. IL-2 secretion correlates primarily with CD28 geometry. (A) Additional geometries of anti-CD3 and anti-CD28 ligands. (B) Six-hour IL-2 secretion on these patterns. Data from each pattern are different from all other conditions (Kruskal–Wallis analysis, {alpha} = 0.05).

 

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