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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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Estimating the kon and koff for sCD80 binding CTLA-4 Ig.  (A) Example of primary data. sCD80 (265 nM) was injected (solid bar) at  40 μl/min through FCs with nothing immobilized (Control) or CTLA-4  immobilized at low (920 RUs) or high (2500 RUs) levels. (B) Effect of  varying the flow rate. sCD80 (265 nM) was injected (solid bar) at 20 (solid  line) or 40 (stippled line) μl/min through FCs with high or low levels of  CTLA-4. Background responses (following injection through a control  FC) have been subtracted. (C) Dissociation of sCD80 from FC with high  (•, ○) or low (▴, ▵) levels of CTLA-4 Ig at flow rate of 20 (•, ▴, ▪)  or 40 (○, ▵, □) μl/min. Also shown is the fall in response in the same  period following injection of sCD80 through control FC (▪, □). The  data fitted reasonably well to single exponential decay curves (dotted lines),  yielding the following t1/2 values: •, 4.8 s; ○, 4.4 s; ▴, 2.2 s; ▵, 2.35 s;  ▪, 0.16 s; □, 0.075 s. (D) Estimating the kon. Equation 1 (see Materials  and Methods) was fitted (solid line) to data (•) from (B) (corresponding to  binding of sCD80 to CTLA-4 [low level] at 40 μl/min), yielding the indicated residuals (▪) and kon. (E) Independent estimation of kon and koff by  analysis of binding at different sCD80 concentrations. The rate at which  the binding rate decreases (−ks) during the injection phase was determined for a range of injected sCD80 concentrations (see Materials and  Methods [43]) using data from Fig. 2 A. A linear regression fit of equation  2 to a plot of ks versus sCD80 concentration yielded the kon and koff values.
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Figure 4: Estimating the kon and koff for sCD80 binding CTLA-4 Ig. (A) Example of primary data. sCD80 (265 nM) was injected (solid bar) at 40 μl/min through FCs with nothing immobilized (Control) or CTLA-4 immobilized at low (920 RUs) or high (2500 RUs) levels. (B) Effect of varying the flow rate. sCD80 (265 nM) was injected (solid bar) at 20 (solid line) or 40 (stippled line) μl/min through FCs with high or low levels of CTLA-4. Background responses (following injection through a control FC) have been subtracted. (C) Dissociation of sCD80 from FC with high (•, ○) or low (▴, ▵) levels of CTLA-4 Ig at flow rate of 20 (•, ▴, ▪) or 40 (○, ▵, □) μl/min. Also shown is the fall in response in the same period following injection of sCD80 through control FC (▪, □). The data fitted reasonably well to single exponential decay curves (dotted lines), yielding the following t1/2 values: •, 4.8 s; ○, 4.4 s; ▴, 2.2 s; ▵, 2.35 s; ▪, 0.16 s; □, 0.075 s. (D) Estimating the kon. Equation 1 (see Materials and Methods) was fitted (solid line) to data (•) from (B) (corresponding to binding of sCD80 to CTLA-4 [low level] at 40 μl/min), yielding the indicated residuals (▪) and kon. (E) Independent estimation of kon and koff by analysis of binding at different sCD80 concentrations. The rate at which the binding rate decreases (−ks) during the injection phase was determined for a range of injected sCD80 concentrations (see Materials and Methods [43]) using data from Fig. 2 A. A linear regression fit of equation 2 to a plot of ks versus sCD80 concentration yielded the kon and koff values.

Mentions: Values for kon and koff were obtained by a linear fit of a plot of ks versus C (see Fig. 4 E).


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)

Estimating the kon and koff for sCD80 binding CTLA-4 Ig.  (A) Example of primary data. sCD80 (265 nM) was injected (solid bar) at  40 μl/min through FCs with nothing immobilized (Control) or CTLA-4  immobilized at low (920 RUs) or high (2500 RUs) levels. (B) Effect of  varying the flow rate. sCD80 (265 nM) was injected (solid bar) at 20 (solid  line) or 40 (stippled line) μl/min through FCs with high or low levels of  CTLA-4. Background responses (following injection through a control  FC) have been subtracted. (C) Dissociation of sCD80 from FC with high  (•, ○) or low (▴, ▵) levels of CTLA-4 Ig at flow rate of 20 (•, ▴, ▪)  or 40 (○, ▵, □) μl/min. Also shown is the fall in response in the same  period following injection of sCD80 through control FC (▪, □). The  data fitted reasonably well to single exponential decay curves (dotted lines),  yielding the following t1/2 values: •, 4.8 s; ○, 4.4 s; ▴, 2.2 s; ▵, 2.35 s;  ▪, 0.16 s; □, 0.075 s. (D) Estimating the kon. Equation 1 (see Materials  and Methods) was fitted (solid line) to data (•) from (B) (corresponding to  binding of sCD80 to CTLA-4 [low level] at 40 μl/min), yielding the indicated residuals (▪) and kon. (E) Independent estimation of kon and koff by  analysis of binding at different sCD80 concentrations. The rate at which  the binding rate decreases (−ks) during the injection phase was determined for a range of injected sCD80 concentrations (see Materials and  Methods [43]) using data from Fig. 2 A. A linear regression fit of equation  2 to a plot of ks versus sCD80 concentration yielded the kon and koff values.
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Figure 4: Estimating the kon and koff for sCD80 binding CTLA-4 Ig. (A) Example of primary data. sCD80 (265 nM) was injected (solid bar) at 40 μl/min through FCs with nothing immobilized (Control) or CTLA-4 immobilized at low (920 RUs) or high (2500 RUs) levels. (B) Effect of varying the flow rate. sCD80 (265 nM) was injected (solid bar) at 20 (solid line) or 40 (stippled line) μl/min through FCs with high or low levels of CTLA-4. Background responses (following injection through a control FC) have been subtracted. (C) Dissociation of sCD80 from FC with high (•, ○) or low (▴, ▵) levels of CTLA-4 Ig at flow rate of 20 (•, ▴, ▪) or 40 (○, ▵, □) μl/min. Also shown is the fall in response in the same period following injection of sCD80 through control FC (▪, □). The data fitted reasonably well to single exponential decay curves (dotted lines), yielding the following t1/2 values: •, 4.8 s; ○, 4.4 s; ▴, 2.2 s; ▵, 2.35 s; ▪, 0.16 s; □, 0.075 s. (D) Estimating the kon. Equation 1 (see Materials and Methods) was fitted (solid line) to data (•) from (B) (corresponding to binding of sCD80 to CTLA-4 [low level] at 40 μl/min), yielding the indicated residuals (▪) and kon. (E) Independent estimation of kon and koff by analysis of binding at different sCD80 concentrations. The rate at which the binding rate decreases (−ks) during the injection phase was determined for a range of injected sCD80 concentrations (see Materials and Methods [43]) using data from Fig. 2 A. A linear regression fit of equation 2 to a plot of ks versus sCD80 concentration yielded the kon and koff values.
Mentions: Values for kon and koff were obtained by a linear fit of a plot of ks versus C (see Fig. 4 E).

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