Altering ICAM-1 mobility perturbs T cell adhesion and priming. (A) ICAM-1^−/− DCs were transduced with GFP or the indicated ICAM-1 constructs, pulsed with peptide at the indicated concentrations, and used to prime CD4⁺ OTII T cells. CD25 surface expression was assessed after 18 h of stimulation. (B) T cells were stimulated as in A and IL-2 secretion was assessed after 18 h using a surface capture assay. (C) CFSE-labeled T cells were stimulated as in A, and CFSE dilution was measured after 96 h to assess proliferation. (D and E) Data were further analyzed to assess the percentage of cells that underwent at least one division (D) and the mean number of divisions of dividing cells (E). Results in A–E are representative of five independent experiments, with data in A, B, D, and E showing mean ± standard deviation (error bars) from triplicate samples in one representative experiment. (F) T cell proliferation was assessed after priming with ICAM-1^−/− DCs or ICAM-1^−/− DCs transduced with WT ICAM-1 or the signaling-incompetent Y518 mutant. Results are representative of three independent experiments. (G) Conjugate formation was assessed by flow cytometry. Data shown are mean ± standard deviation (error bars) from triplicate samples in one experiment, representative of four individual experiments. (H) Representative midplane images showing T cells interacting with DCs that do or do not display capped ICAM-1. Bars, 10 µm. (I) Conjugates were prepared and imaged as in F. DCs interacting with two T cells were randomly selected and scored for homotypic T cell interactions. Data represent means ± standard deviation (error bars) from four independent experiments, with at least 50 cells each. *, P < 0.05.