Limits...
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.

Show MeSH

Related in: MedlinePlus

Estimating the kon and koff for sCD80 binding CD28 Ig. (A)  Example of primary data. sCD80 (2.65 μM) was injected (solid bar) at 80  μl/min through FCs with nothing immobilized (Control) or CD28 Ig immobilized at low (3400 RUs) or high (6200 RUs) levels. (B) Effect of  varying the flow rate. sCD80 (2.65 μM) was injected (solid bar) at 40 (solid  line) or 80 (stippled line) μl/min through FCs with high or low levels of  CD28 Ig. Background responses (following injection through a control  FC) have been subtracted. (C) Dissociation of sCD80 from FC with high  (•, ○) or low (▴, ▵) levels of CD28 Ig at flow rate of 40 (•, ▴, ▪) or  80 (○, ▵, □) μl/min. Also shown is the fall in response in the same period following injection of sCD80 through a control FC (▪, □). The data  fitted well to single exponential decay curves (dotted lines), yielding the  following t1/2 values: •, 0.93 s; ○, 0.84 s; ▴, 0.69 s; Δ, 0.64 s; ▪, 0.075 s;  □, 0.04 s. (D) Obtaining the kon by nonlinear curve fitting. Eq. 1 (see  Materials and Methods) was fitted (solid line) to data (•) from (B) (corresponding to binding of sCD80 to CD28 Ig [low level] at 80 μl/min),  yielding the indicated residuals (▪) and kon.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196039&req=5

Figure 5: Estimating the kon and koff for sCD80 binding CD28 Ig. (A) Example of primary data. sCD80 (2.65 μM) was injected (solid bar) at 80 μl/min through FCs with nothing immobilized (Control) or CD28 Ig immobilized at low (3400 RUs) or high (6200 RUs) levels. (B) Effect of varying the flow rate. sCD80 (2.65 μM) was injected (solid bar) at 40 (solid line) or 80 (stippled line) μl/min through FCs with high or low levels of CD28 Ig. Background responses (following injection through a control FC) have been subtracted. (C) Dissociation of sCD80 from FC with high (•, ○) or low (▴, ▵) levels of CD28 Ig at flow rate of 40 (•, ▴, ▪) or 80 (○, ▵, □) μl/min. Also shown is the fall in response in the same period following injection of sCD80 through a control FC (▪, □). The data fitted well to single exponential decay curves (dotted lines), yielding the following t1/2 values: •, 0.93 s; ○, 0.84 s; ▴, 0.69 s; Δ, 0.64 s; ▪, 0.075 s; □, 0.04 s. (D) Obtaining the kon by nonlinear curve fitting. Eq. 1 (see Materials and Methods) was fitted (solid line) to data (•) from (B) (corresponding to binding of sCD80 to CD28 Ig [low level] at 80 μl/min), yielding the indicated residuals (▪) and kon.

Mentions: Measurements of binding kinetics are prone to error, particularly when the kinetics are very fast, as in the present study (48–50). One source of error is that part of the response is a background signal that does not represent true binding. To correct for this, the response obtained when sCD80 was injected through a control flow cell (containing no immobilized protein) was subtracted before analysis. Figs. 4 A and 5 A show typical responses obtained after injection of sCD80 through flow cells with two different levels of CTLA-4 Ig (or CD28 Ig) immobilized, as well as through a control flow cell. Subtraction of the control flow cell response from the responses in the CD28 and CTLA-4 flow cells gives the actual binding response shown in Figs. 4 B and 5 B (solid lines), which was subjected to kinetic analysis. A second potential source of error is that binding and dissociation are limited by the rate at which protein is delivered to and removed from the sensor surface. This was addressed by using higher than normal flow rates (40 or 80 μl/min). These flow rates were judged to be optimal because halving them had little effect on the association (injection) or dissociation (washout) phases when sCD80 was injected over CTLA-4 (see Figs. 4 B and C) or CD28 (Figs. 5, B and C). It should be noted that even at very high flow rates, binding may still be limited by mass-transport within an unstirred layer close to the binding surface (48, 50). The effects of mass-transport limitations within this unstirred layer can be reduced by decreasing the level of immobilized ligand (see below). A third source of error is rebinding of protein during the dissociation (washout) phase, which will lead to underestimation of the dissociation rate. Rebinding can be decreased by immobilizing lower levels of ligand. This is illustrated in Figs. 4 C and 5 C, in which sCD80 dissociates more rapidly when the level of immobilized CTLA-4 or CD28 is decreased. sCD80 dissociated from sensor surfaces with high and low levels of CTLA-4 with apparent dissociation rate constants (koff) of 0.24 s−1 and 0.43 s−1, respectively (see Fig. 4 C; Table 3). Similarly, sCD80 dissociated from high and low levels of CD28 with koff values of 1.1 s−1 and 1.6 s−1, respectively (Fig. 5 C; Table 3). Although it is quite likely that some sCD80 rebinding is still occurring even at these lower CD28 Ig and CTLA-4 Ig levels, it proved difficult to collect data of satisfactory quality when CD28 Ig and CTLA-4 Ig levels were reduced further. Therefore, we assume that the actual koff values for dissociation of sCD80 from CTLA-4 and CD28 are at least 0.43 s−1 and 1.6 s−1, respectively.


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 CD28 Ig. (A)  Example of primary data. sCD80 (2.65 μM) was injected (solid bar) at 80  μl/min through FCs with nothing immobilized (Control) or CD28 Ig immobilized at low (3400 RUs) or high (6200 RUs) levels. (B) Effect of  varying the flow rate. sCD80 (2.65 μM) was injected (solid bar) at 40 (solid  line) or 80 (stippled line) μl/min through FCs with high or low levels of  CD28 Ig. Background responses (following injection through a control  FC) have been subtracted. (C) Dissociation of sCD80 from FC with high  (•, ○) or low (▴, ▵) levels of CD28 Ig at flow rate of 40 (•, ▴, ▪) or  80 (○, ▵, □) μl/min. Also shown is the fall in response in the same period following injection of sCD80 through a control FC (▪, □). The data  fitted well to single exponential decay curves (dotted lines), yielding the  following t1/2 values: •, 0.93 s; ○, 0.84 s; ▴, 0.69 s; Δ, 0.64 s; ▪, 0.075 s;  □, 0.04 s. (D) Obtaining the kon by nonlinear curve fitting. Eq. 1 (see  Materials and Methods) was fitted (solid line) to data (•) from (B) (corresponding to binding of sCD80 to CD28 Ig [low level] at 80 μl/min),  yielding the indicated residuals (▪) and kon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196039&req=5

Figure 5: Estimating the kon and koff for sCD80 binding CD28 Ig. (A) Example of primary data. sCD80 (2.65 μM) was injected (solid bar) at 80 μl/min through FCs with nothing immobilized (Control) or CD28 Ig immobilized at low (3400 RUs) or high (6200 RUs) levels. (B) Effect of varying the flow rate. sCD80 (2.65 μM) was injected (solid bar) at 40 (solid line) or 80 (stippled line) μl/min through FCs with high or low levels of CD28 Ig. Background responses (following injection through a control FC) have been subtracted. (C) Dissociation of sCD80 from FC with high (•, ○) or low (▴, ▵) levels of CD28 Ig at flow rate of 40 (•, ▴, ▪) or 80 (○, ▵, □) μl/min. Also shown is the fall in response in the same period following injection of sCD80 through a control FC (▪, □). The data fitted well to single exponential decay curves (dotted lines), yielding the following t1/2 values: •, 0.93 s; ○, 0.84 s; ▴, 0.69 s; Δ, 0.64 s; ▪, 0.075 s; □, 0.04 s. (D) Obtaining the kon by nonlinear curve fitting. Eq. 1 (see Materials and Methods) was fitted (solid line) to data (•) from (B) (corresponding to binding of sCD80 to CD28 Ig [low level] at 80 μl/min), yielding the indicated residuals (▪) and kon.
Mentions: Measurements of binding kinetics are prone to error, particularly when the kinetics are very fast, as in the present study (48–50). One source of error is that part of the response is a background signal that does not represent true binding. To correct for this, the response obtained when sCD80 was injected through a control flow cell (containing no immobilized protein) was subtracted before analysis. Figs. 4 A and 5 A show typical responses obtained after injection of sCD80 through flow cells with two different levels of CTLA-4 Ig (or CD28 Ig) immobilized, as well as through a control flow cell. Subtraction of the control flow cell response from the responses in the CD28 and CTLA-4 flow cells gives the actual binding response shown in Figs. 4 B and 5 B (solid lines), which was subjected to kinetic analysis. A second potential source of error is that binding and dissociation are limited by the rate at which protein is delivered to and removed from the sensor surface. This was addressed by using higher than normal flow rates (40 or 80 μl/min). These flow rates were judged to be optimal because halving them had little effect on the association (injection) or dissociation (washout) phases when sCD80 was injected over CTLA-4 (see Figs. 4 B and C) or CD28 (Figs. 5, B and C). It should be noted that even at very high flow rates, binding may still be limited by mass-transport within an unstirred layer close to the binding surface (48, 50). The effects of mass-transport limitations within this unstirred layer can be reduced by decreasing the level of immobilized ligand (see below). A third source of error is rebinding of protein during the dissociation (washout) phase, which will lead to underestimation of the dissociation rate. Rebinding can be decreased by immobilizing lower levels of ligand. This is illustrated in Figs. 4 C and 5 C, in which sCD80 dissociates more rapidly when the level of immobilized CTLA-4 or CD28 is decreased. sCD80 dissociated from sensor surfaces with high and low levels of CTLA-4 with apparent dissociation rate constants (koff) of 0.24 s−1 and 0.43 s−1, respectively (see Fig. 4 C; Table 3). Similarly, sCD80 dissociated from high and low levels of CD28 with koff values of 1.1 s−1 and 1.6 s−1, respectively (Fig. 5 C; Table 3). Although it is quite likely that some sCD80 rebinding is still occurring even at these lower CD28 Ig and CTLA-4 Ig levels, it proved difficult to collect data of satisfactory quality when CD28 Ig and CTLA-4 Ig levels were reduced further. Therefore, we assume that the actual koff values for dissociation of sCD80 from CTLA-4 and CD28 are at least 0.43 s−1 and 1.6 s−1, respectively.

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