Coating with CCL21+ICAM1 increases T-cell proliferation following antigen-specific and nonspecific activation, whereas IL-6 increases cell survival. (A-D) Representative images of OT-II T cells expressing ubiquitin-green fluorescent protein (green) grown in the different indicated conditions for 7 days, after the breaking down of the clusters, and their spin-down. Dead cell nuclei are stained in red. Scale bars, 50 μm. (E-F) Bar graphs illustrating the percentage of dead cells (E) and the number of viable cells (F), as quantified using automated image analysis (number of T cells seeded per well: 12 × 103; data are representative of at least 3 independent experiments with 5 replicates (wells) and 20 fields of view in each well (see supplemental Figure 3 for summary of all independent expansion tests). Error bars represent standard error of the mean. IL-6 decreased the percentage of dead cells by approximately twofold on both coated and uncoated surfaces (E). IL-6 increased live cell numbers by 7.7-fold when combined with the CCL21+ICAM1 coating. CCL21+ICAM1 coating with no IL-6 increased cell numbers by 4.9-fold compared with the uncoated surface (F). (G-H) Histogram and bar graph illustrating the increase in cell proliferation induced by CCL21+ICAM1 with or without IL-6 following antigen-specific activation, as measured by CFSE dilution (data are representative of at least 3 independent experiments with 3 replicates each; error bars represent standard error of the mean). Color code in panel G: green, nonactivated T cells; red, T cells activated without coating; purple, T cells activated on CCL21+ICAM1 coating; orange, T cells activated on CCL21+ICAM1 coating with IL-6. (I) Bar graph illustrating live cell number, measured using a metabolic cell viability assay (data are representative of at least 3 independent experiments with 4 replicates each; error bars represent standard error of the mean) of T cells activated with either antigen-loaded DCs or activation beads, with or without CCL21+ICAM1 coating. Number of T cells seeded per well was 60 × 103. CCL21+ICAM1 increases T-cell yield in both activation methods. (J-M) Representative images demonstrating higher cell densities in cultures with CCL21+ICAM1 compared with no coating, activated with either activation beads (J-K) or antigen-loaded DCs (L-M). Scale bars, 50 μm. (N-O) Histogram and bar graph illustrating the increase in cell proliferation induced by CCL21+ICAM1 with and without IL-6, following activation with beads, as measured by CFSE dilution (data are representative of at least 3 independent experiments with 3 replicates each; error bars represent standard error of the mean). Color code in panel N is the same as that described in panel G. Calculated P values in E-F, H-I, and O (using t test) are as indicated in the figures.
