Kinetics of MHC-CD8 Interaction at the T Cell Membrane

Abstract

CD8 plays an important role in facilitating TCR-MHC interaction, promoting Ag recognition, and initiating T cell activation. MHC-CD8 binding kinetics have been measured in three dimensions by surface plasmon resonance technique using purified molecules. However, CD8 is a membrane-anchored, signaling kinase-linked, and TCR-associated molecule whose function depends on the cell membrane environment. Purified molecules lack their linkage to the membrane, which precludes interactions with other structures of the cell as well as signaling. Furthermore, three-dimensional binding in the fluid phase is biologically and physically distinct from two-dimensional binding across apposing cell membranes. As a first step toward characterizing the molecular interactions between T cells and APCs, we used a micropipette adhesion frequency assay to measure the adhesion kinetics of single mouse T cells interacting with single human RBCs coated with MHC. Using anti-TCR mAb we isolated and characterized the specific two-dimensional MHC-CD8 binding from the trimolecular TCR-MHC-CD8 interaction. The TCR-independent MHC-CD8 interaction has a very low affinity that depends on the MHC alleles, but not on the peptide complexed to the MHC and whether CD8 is an αα homodimer or an αβ heterodimer. Surprisingly, MHC-CD8 binding affinity varies with T cells from different TCR transgenic mice and these affinity differences were abolished by treatment with cholesterol oxidase to disrupt membrane rafts. These data highlight the relevance and importance of two-dimensional analysis of T cells and APCs and indicate that membrane rafts play an important role in modulating the affinity of cell-cell interactions.

Recognition of Class I MHC by a Rat Ly49 NK Cell Receptor Is Dependent on the Identity of the P2 Anchor Amino Acid of Bound Peptide

“…Our results indicate that Ly49i2 is peptide selective in MHC-I binding, such that a subset of RT1-A1 c molecules, perhaps only those with a Pro or Val in the P2 position of bound peptide, are recognized by Ly49i2. In contrast, mouse CD8 is not peptide selective in its interaction with MHC-I ligands (50,51), as is also likely for rat CD8. Thus, there would likely be some RT1-A1 c molecules on the surface of a potential target cell that are ligands for either Ly49i2 or CD8 and others that are ligands only for CD8.…”

Adhesion Frequency Assay for <em>In Situ</em> Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

“…These include the membrane microtopology 5 , membrane anchor 2 , molecular orientation and length 6 , carrier stiffness 9 and curvature 20 , impingement force 20 , and modulators of the cytoskeleton and membrane organization where the interacting molecules reside 15,17 .…”

“…The assay has been validated using interactions of Fcγ receptors with IgG Fc 1-6 , selectins with glycoconjugate ligands 6-9 , integrins with ligands 10-13 , homotypical cadherin binding 14 , T cell receptor and coreceptor with peptide-major histocompatibility complexes [15][16][17][18][19] .…”

“…The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology 5 , membrane anchor 2 , molecular orientation and length 6 , carrier stiffness 9 , curvature 20 , and impingement force 20 , as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules 15,17,19 .…”

“…Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics 1 to the binding curve returns the 2D affinity and off-rate.The assay has been validated using interactions of Fcγ receptors with IgG Fc 1-6 , selectins with glycoconjugate ligands 6-9 , integrins with ligands 10-13 , homotypical cadherin binding 14 , T cell receptor and coreceptor with peptide-major histocompatibility complexes [15][16][17][18][19] .The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology 5 , membrane anchor 2 , molecular orientation and length 6 , carrier stiffness 9 , curvature 20 , and impingement force 20 , as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules 15,17,19 .The method has also been used to study the concurrent binding of dual receptor-ligand species 3,4 , and trimolecular interactions 19 using a modified model 21 .The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors 17 .…”