Figure 6. Restriction of ICAM-1 lateral... | Rockefeller University Press

Figure 6.

$Figure 6. Restriction of ICAM-1 lateral mobility promotes LFA-1 affinity maturation. (A, top) Diagram of LFA-1 conformational states with conformation-specific mAb binding sites. (A, bottom) Schematic showing 293T cell–based artificial APCs used to stimulate T cells, as detailed in the Materials and methods. (B and C) Mobile fraction (B) and diffusion coefficient (C) of ICAM-1 in 293T artificial APCs. Dots represent individual FRAP measurements (n = 140–215) pooled from three independent experiments. (D) Ex vivo human T cells were allowed to interact with artificial APCs lacking ICAM-1 (null) or transduced with WT ICAM-1. Conjugates were fixed and labeled with conformation-specific anti–LFA-1 antibodies. Representative micrographs are shown. Bars, 10 µm. (E and F) Conjugates were prepared as in D. (E) The relative proportion of LFA-1 in the extended conformation was assessed based on the ratio of Kim127:TS2/4 labeling intensity. (F) The relative proportion of LFA-1 in the extended open conformation was assessed based on the ratio of m24:TS2/4 labeling intensity. (G–I) Conjugates were prepared and analyzed as in D–F, except that resting T lymphoblasts were used. H and I show T cells from two different human donors in order to show reproducibility. Dots in E–I represent values from single cells (n = 22–107 cells per condition) pooled from two independent experiments (E and F) or three independent experiments (G–I). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.$

Restriction of ICAM-1 lateral mobility promotes LFA-1 affinity maturation. (A, top) Diagram of LFA-1 conformational states with conformation-specific mAb binding sites. (A, bottom) Schematic showing 293T cell–based artificial APCs used to stimulate T cells, as detailed in the Materials and methods. (B and C) Mobile fraction (B) and diffusion coefficient (C) of ICAM-1 in 293T artificial APCs. Dots represent individual FRAP measurements (n = 140–215) pooled from three independent experiments. (D) Ex vivo human T cells were allowed to interact with artificial APCs lacking ICAM-1 (null) or transduced with WT ICAM-1. Conjugates were fixed and labeled with conformation-specific anti–LFA-1 antibodies. Representative micrographs are shown. Bars, 10 µm. (E and F) Conjugates were prepared as in D. (E) The relative proportion of LFA-1 in the extended conformation was assessed based on the ratio of Kim127:TS2/4 labeling intensity. (F) The relative proportion of LFA-1 in the extended open conformation was assessed based on the ratio of m24:TS2/4 labeling intensity. (G–I) Conjugates were prepared and analyzed as in D–F, except that resting T lymphoblasts were used. H and I show T cells from two different human donors in order to show reproducibility. Dots in E–I represent values from single cells (n = 22–107 cells per condition) pooled from two independent experiments (E and F) or three independent experiments (G–I). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.