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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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Comparison of avidity of CD28 Ig and CTLA-4 Ig binding  to immobilized sCD80. CD28 Ig or CTLA-4 Ig were injected at 5 μl/ min (25°C) at the indicated concentration for 6–8 min through FC with  either sCD80 (3300 RU) or no protein (Control) immobilized. The responses seen with injection of the three CTLA4 Ig concentrations  through the Control FC were indistinguishable when superimposed.
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Figure 6: Comparison of avidity of CD28 Ig and CTLA-4 Ig binding to immobilized sCD80. CD28 Ig or CTLA-4 Ig were injected at 5 μl/ min (25°C) at the indicated concentration for 6–8 min through FC with either sCD80 (3300 RU) or no protein (Control) immobilized. The responses seen with injection of the three CTLA4 Ig concentrations through the Control FC were indistinguishable when superimposed.

Mentions: The affinities measured in the present study for CD80 binding CD28 (Kd 4 μM) and CTLA-4 (Kd 0.42 μM) are much lower than previously reported (8, 29). One possible difference is that these earlier measurements were made at 23°C (8, 29). However, we found that decreasing the temperature from 37°C to 25°C increased the affinity less than twofold (Table 2). A more significant difference is likely to be that the soluble CD80 Ig used in the earlier studies was not monomeric. This is supported by the observation that the CD80 Ig fusion protein used in these earlier studies, although monomeric on SDS-PAGE (Mr 70,000), eluted at Mr ∼350,000 by size-exclusion chromatography (29), suggesting the presence of higher aggregates. Such aggregates could well account for the much higher affinities previously reported. However, this explanation does not account for 100- to 200-fold higher avidity observed for soluble CTLA-4 Ig compared with soluble CD28 Ig (for example see reference 30), a difference much larger than the 10-fold difference in affinity reported in the present study. This discrepancy is not due to differences in the recombinant proteins used in these studies, because we were able to reproduce the large difference in avidity by injecting CD28 Ig and CTLA4 Ig over a sensor surface onto which sCD80 had been immobilized (Fig. 6). CTLA-4 Ig injected at 0.1 and 1 μg/ml bound to higher levels than 100-fold higher concentrations of CD28 Ig (Fig. 6), indicating a >100-fold higher avidity. Although it is unclear why the avidity difference between CTLA-4 Ig and CD28 Ig is so much greater than the affinity difference, these results illustrate a major disadvantage of using multimeric proteins for comparative binding studies.


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)

Comparison of avidity of CD28 Ig and CTLA-4 Ig binding  to immobilized sCD80. CD28 Ig or CTLA-4 Ig were injected at 5 μl/ min (25°C) at the indicated concentration for 6–8 min through FC with  either sCD80 (3300 RU) or no protein (Control) immobilized. The responses seen with injection of the three CTLA4 Ig concentrations  through the Control FC were indistinguishable when superimposed.
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Related In: Results  -  Collection

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Figure 6: Comparison of avidity of CD28 Ig and CTLA-4 Ig binding to immobilized sCD80. CD28 Ig or CTLA-4 Ig were injected at 5 μl/ min (25°C) at the indicated concentration for 6–8 min through FC with either sCD80 (3300 RU) or no protein (Control) immobilized. The responses seen with injection of the three CTLA4 Ig concentrations through the Control FC were indistinguishable when superimposed.
Mentions: The affinities measured in the present study for CD80 binding CD28 (Kd 4 μM) and CTLA-4 (Kd 0.42 μM) are much lower than previously reported (8, 29). One possible difference is that these earlier measurements were made at 23°C (8, 29). However, we found that decreasing the temperature from 37°C to 25°C increased the affinity less than twofold (Table 2). A more significant difference is likely to be that the soluble CD80 Ig used in the earlier studies was not monomeric. This is supported by the observation that the CD80 Ig fusion protein used in these earlier studies, although monomeric on SDS-PAGE (Mr 70,000), eluted at Mr ∼350,000 by size-exclusion chromatography (29), suggesting the presence of higher aggregates. Such aggregates could well account for the much higher affinities previously reported. However, this explanation does not account for 100- to 200-fold higher avidity observed for soluble CTLA-4 Ig compared with soluble CD28 Ig (for example see reference 30), a difference much larger than the 10-fold difference in affinity reported in the present study. This discrepancy is not due to differences in the recombinant proteins used in these studies, because we were able to reproduce the large difference in avidity by injecting CD28 Ig and CTLA4 Ig over a sensor surface onto which sCD80 had been immobilized (Fig. 6). CTLA-4 Ig injected at 0.1 and 1 μg/ml bound to higher levels than 100-fold higher concentrations of CD28 Ig (Fig. 6), indicating a >100-fold higher avidity. Although it is unclear why the avidity difference between CTLA-4 Ig and CD28 Ig is so much greater than the affinity difference, these results illustrate a major disadvantage of using multimeric proteins for comparative binding studies.

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