CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics
van der Merwe PA, Bodian DL, Daenke S, Linsley P, Davis SJ. (1997), J Exp Med. 185, 393-403
The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values ∼12 and ∼200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell–APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37°C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 μM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (koff); sCD80 dissociated from CD28 and CTLA-4 with koff values of ⩾1.6 and ⩾0.43 s−1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate dynamic T cell–APC contacts and to facilitate scanning of APC for antigen.
Measuring the affinity of sCD80 binding to CTLA-4 Ig by equilibrium binding. (A) A range of sCD80 concentrations (2.65 μM and nine twofold dilutions thereof) were injected sequentially (solid bar) for 2 min at 10 μl/min through a flow cell (FC) with either CTLA-4 Ig (1,500 RUs) or no protein (Control) immobilized. sCD80 (0.26 μM) bound to a very similar level (255 and 250 RUs) when injected at the beginning and end of the experiment (data not shown), indicating that the immobilized CTLA-4 Ig was stable. (B) The equilibrium responses in the CTLA-4 Ig (•) and Control (▴) FCs at each sCD80 concentration and the differences between these responses (representing actual binding, ▪) are plotted. The dotted line represents a nonlinear fit of the Langmuir binding isotherm to the binding data and yields a Kd of 0.4 μM and a binding maximum of 774 RUs. A Scatchard plot of the same data is shown on the right. A linear regression fit yields a Kd of 0.4 μM and a binding maximum of 750 RUs.