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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 - J. Exp. Med. (1997)

Bottom Line: Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 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 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, United Kingdom.

ABSTRACT
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 approximately 12 and approximately 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 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 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.

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Measuring the affinity of sCD80 binding to CD28 Ig by  equilibrium binding. (A) A range of sCD80 concentrations (26.5 μM and  eight twofold dilutions thereof) were injected sequentially (solid bar) for  30 s at 10 μl/min through a FC with either CD28 Ig (immobilized at a  level of 7800 RUs) or an irrelevant protein (Control) immobilized. sCD80  (0.82 μM) injected at the beginning and end of the experiment bound to  a very similar level (160 and 157 RUs), indicating that the immobilized  CD28 Ig was stable. (B) The equilibrium responses in the CD28 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 4.1 μM and a binding maximum of  939 RUs. A Scatchard plot of the same data is shown on the right. A linear  regression fit yields a Kd of 4.3 μM and a binding maximum of 952 RUs.
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Figure 3: Measuring the affinity of sCD80 binding to CD28 Ig by equilibrium binding. (A) A range of sCD80 concentrations (26.5 μM and eight twofold dilutions thereof) were injected sequentially (solid bar) for 30 s at 10 μl/min through a FC with either CD28 Ig (immobilized at a level of 7800 RUs) or an irrelevant protein (Control) immobilized. sCD80 (0.82 μM) injected at the beginning and end of the experiment bound to a very similar level (160 and 157 RUs), indicating that the immobilized CD28 Ig was stable. (B) The equilibrium responses in the CD28 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 4.1 μM and a binding maximum of 939 RUs. A Scatchard plot of the same data is shown on the right. A linear regression fit yields a Kd of 4.3 μM and a binding maximum of 952 RUs.

Mentions: Affinity and kinetic measurements were performed at 37°C except where otherwise indicated. The affinity of sCD80 binding to CTLA-4 and CD28 was measured directly by equilibrium binding analysis (Figs. 2 and 3), because this avoids the many potential pitfalls associated with kinetic measurements (see below; 48–50). Increasing concentrations of sCD80 were injected over sensor surfaces to which CTLA-4 Ig or CD28 Ig had been immobilized (Figs. 2 A and 3 A, respectively). It is noteworthy that at all sCD80 concentrations binding reached equilibrium very rapidly (95% binding within 20 s; faster for CD28) and that, in the washout phase after the injection, bound sCD80 dissociated within 20 s (faster for CD28). These features are typical of interactions with very fast binding kinetics. The sCD80 samples were also injected over a control sensor surface (with no protein immobilized) to measure the background response (Figs. 2 A and 3 A, right). Larger background responses are seen in Fig. 3 A because a tenfold higher range of sCD80 concentrations was injected over CD28 Ig (Figs. 2 and 3, legends). For each sCD80 concentration the binding response (measured in arbitrary response units [RU]) at equilibrium was calculated by subtracting the response seen in the control flow cell from the response seen in the CTLA-4 (see Fig. 2 B) or CD28 (Fig. 3 B) flow cells. A plot of the binding response indicates saturable binding and direct fitting of a standard Langmuir binding isotherm to the data gave Kd values of 0.38 and 4.2 μM for sCD80 binding to CTLA-4 and CD28, respectively (Figs. 2 B and 3 B, left). Scatchard plots of the data gave very similar Kd values (Figs. 2 B and 3 B, right). The mean Kd (± SD) values from several independent determinations are shown in Table 2.


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 - J. Exp. Med. (1997)

Measuring the affinity of sCD80 binding to CD28 Ig by  equilibrium binding. (A) A range of sCD80 concentrations (26.5 μM and  eight twofold dilutions thereof) were injected sequentially (solid bar) for  30 s at 10 μl/min through a FC with either CD28 Ig (immobilized at a  level of 7800 RUs) or an irrelevant protein (Control) immobilized. sCD80  (0.82 μM) injected at the beginning and end of the experiment bound to  a very similar level (160 and 157 RUs), indicating that the immobilized  CD28 Ig was stable. (B) The equilibrium responses in the CD28 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 4.1 μM and a binding maximum of  939 RUs. A Scatchard plot of the same data is shown on the right. A linear  regression fit yields a Kd of 4.3 μM and a binding maximum of 952 RUs.
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Related In: Results  -  Collection

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Figure 3: Measuring the affinity of sCD80 binding to CD28 Ig by equilibrium binding. (A) A range of sCD80 concentrations (26.5 μM and eight twofold dilutions thereof) were injected sequentially (solid bar) for 30 s at 10 μl/min through a FC with either CD28 Ig (immobilized at a level of 7800 RUs) or an irrelevant protein (Control) immobilized. sCD80 (0.82 μM) injected at the beginning and end of the experiment bound to a very similar level (160 and 157 RUs), indicating that the immobilized CD28 Ig was stable. (B) The equilibrium responses in the CD28 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 4.1 μM and a binding maximum of 939 RUs. A Scatchard plot of the same data is shown on the right. A linear regression fit yields a Kd of 4.3 μM and a binding maximum of 952 RUs.
Mentions: Affinity and kinetic measurements were performed at 37°C except where otherwise indicated. The affinity of sCD80 binding to CTLA-4 and CD28 was measured directly by equilibrium binding analysis (Figs. 2 and 3), because this avoids the many potential pitfalls associated with kinetic measurements (see below; 48–50). Increasing concentrations of sCD80 were injected over sensor surfaces to which CTLA-4 Ig or CD28 Ig had been immobilized (Figs. 2 A and 3 A, respectively). It is noteworthy that at all sCD80 concentrations binding reached equilibrium very rapidly (95% binding within 20 s; faster for CD28) and that, in the washout phase after the injection, bound sCD80 dissociated within 20 s (faster for CD28). These features are typical of interactions with very fast binding kinetics. The sCD80 samples were also injected over a control sensor surface (with no protein immobilized) to measure the background response (Figs. 2 A and 3 A, right). Larger background responses are seen in Fig. 3 A because a tenfold higher range of sCD80 concentrations was injected over CD28 Ig (Figs. 2 and 3, legends). For each sCD80 concentration the binding response (measured in arbitrary response units [RU]) at equilibrium was calculated by subtracting the response seen in the control flow cell from the response seen in the CTLA-4 (see Fig. 2 B) or CD28 (Fig. 3 B) flow cells. A plot of the binding response indicates saturable binding and direct fitting of a standard Langmuir binding isotherm to the data gave Kd values of 0.38 and 4.2 μM for sCD80 binding to CTLA-4 and CD28, respectively (Figs. 2 B and 3 B, left). Scatchard plots of the data gave very similar Kd values (Figs. 2 B and 3 B, right). The mean Kd (± SD) values from several independent determinations are shown in Table 2.

Bottom Line: Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values approximately 12 and approximately 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 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, United Kingdom.

ABSTRACT
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 approximately 12 and approximately 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 degrees C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 microM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (k(off)); sCD80 dissociated from CD28 and CTLA-4 with k(off) values of > or = 1.6 and > or = 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.

Show MeSH
Related in: MedlinePlus