TCR signaling can enhance the avidity of CD28 ligand binding.

(A) Images from a cell adhesion assay. Top, a resting T cell and CD80-coated bead brought into contact using micropipets. Middle, the same T cell 30s later after the cell and bead were separated. No adhesion was observed. Bottom, image of a T cell bound to an anti-CD3 coated bead, which is out of the plane of focus, but clearly distal to the site of interaction of the T cell with the CD80-bead. This image was taken shortly after the T cell was pulled away from the CD80-bead. In this case, the T cell-CD80-bead adhesion has caused the T cell to dislodge from the micropipet. (B) Cell frequency adhesion of in vitro primed and resting WT DO11.10 T cells to control beads (None) or beads coated with CD80 (CD80-Fc) in the absence (Unstim, empty bars) and after (Anti-CD3; filled bars) stimulation. No increase in adhesion to CD80 was detected when cells were stimulated with beads coated with anti-MHC class I (not shown). (C and D) WT and CD28-deficient T cells were primed with CD80/CD86-negative APC to reduce upregulation of CTLA-4. (C) Flow cytometry of CTLA-4 expression on WT (thin line) and CD28-deficient (thick line) T cells after in vitro priming. Isotype control (filled) and WT T cells primed in the presence of CD28 costimulation (dashed line) are included as negative and positive controls. (D) Cell frequency adhesion of WT and CD28-deficient (CD28KO) T cells that were primed with CD80/CD86-negative cells, to CD80-beads in the absence (Unstim) and after (Anti-CD3) stimulation. (E) Adhesion frequency of WT and CTLA-4-deficient (CTLA-4KO) T cells to control beads (None) or beads coated with increasing concentrations of CD80 in the absence (Unstim) and after (Anti-CD3) stimulation. Cell adhesion data are presented as mean ± SD of the adhesion frequency for individual cells (25 impingements each; n = 9–10, except for 62.5 ng/ml CD80-Fc in panel E, n = 6; p values for t tests between samples are shown; ns, not significant).