Funding

NIH Grant

National Institutes of Health

Cell-cell adhesion plays a crucial role in the function of the immune system. The formation of a stable conjugate between a T lymphocyte and an antigen-presenting cell (APC) is a pre-requisite for T cell activation and is mediated by the interaction between lymphocyte function-associated antigen 1 (LFA 1) and intercellular adhesion molecule 1 (ICAM 1). Although it has been shown that T lymphocytes can modulate the LFA 1/ICAM 1 interaction, it is not known how changes in the affinity state of LFA-1 translate to changes in the mechanical strength of the LFA 1/ICAM 1 bond. Moreover, it is not known how the lateral redistribution of LFA 1 following cell activation modifies the strength of cell-cell adhesion. This gap in knowledge limits the extent to which an effective strategy can be developed to regulate the immune response through the modulation of cell-cell adhesion. Our long-range goal is to understand the mechanisms that regulate leukocyte adhesion during immune and inflammatory processes. The main objective of the proposed research is to determine the biophysical parameters that enable the LFA 1/ICAM 1 interaction to facilitate firm adhesion between T lymphocytes and APCs. The central hypothesis of this application is that cell adhesion is governed by the intrinsic properties of the LFA 1/ICAM 1 complex and by the ability of the complex to modulate between different adhesive states. We have formulated this hypothesis based on preliminary findings that (i) the LFA 1/ICAM 1 complex is especially well-suited to resist large pulling forces and (ii) the avidity modulation of LFA-1 plays an important role in promoting enhanced cell adhesion. We plan to test our hypothesis and accomplish the objectives of this application by pursuing the following specific aims:

Aim 1: Characterize the dissociation pathway of the human LFA 1/ICAM 1 complex to determine how this complex responds to a pulling force.

The goal of this aim is to determine how the high and low affinity LFA 1/ICAM 1 complexes unbind under the influence of a pulling force. The working hypothesis is that the unbinding of the LFA 1/ICAM 1 complex involves overcoming multiple energy barriers before final separation. Using atomic force microscopy (AFM), each of these energy barriers can be characterized by measuring the unbinding (or rupture) force of the complex at different loading rate regimes. We propose to extend the dynamic range and resolution of the force spectra of the LFA 1/ICAM 1 interaction to faster loading rates in order to obtain a detailed description of the dissociation pathway. To achieve this objective, we will carry out the force measurements in a cell-free system to reduce non-specific interactions between the interacting surfaces and to achieve faster loading rates. The proposed measurements will reveal how LFA-1 activation changes the dissociation pathway of the LFA 1/ICAM-1 complex.

Aim 2: Determine how conformational changes in the LFA-1 I domain strengthen the LFA 1/ICAM 1 interaction.

The transition between the high and low affinity states of the LFA 1/ICAM 1 interaction has been attributed to the expression of different LFA 1 conformers. Recent studies have correlated LFA 1 affinity to structural changes in the I domain of the  subunit of LFA 1. Locking the I domain in an “open” or “closed” conformation by genetically designed disulfide linkages resulted in a constitutively high or low affinity conformer of LFA 1. Similarly, certain site-directed mutations in the LFA 1 I domain were able to stabilize the high or low affinity conformer. In this project, we propose to determine the relationship between the different conformers of LFA 1 and the dynamic strength of the LFA 1/ICAM 1 complex by AFM.

Aim 3: Identify the molecular determinants that give the LFA 1/ICAM 1 complex its mechanical strength.

Our working hypothesis is that the dissociation of the LFA 1/ICAM 1 complex involves overcoming two activation energy barriers: an inner barrier and an outer barrier. We attribute the inner barrier to the strong electrostatic interaction between the chelated cation in the I domain of LFA-1 and Glu 34 of ICAM 1 and the outer barrier to the interactions of the surrounding complementary binding surfaces. In the proposed research, selected amino acid residues on the predicted binding surface of both LFA 1 and ICAM 1 will be mutated to determine their roles in stabilizing the LFA 1/ICAM 1 complex. Subsequent acquisition and analysis of the force spectra (i.e., unbinding force vs. loading rate relation) from mutated LFA 1/ICAM 1 complexes will identify the molecular determinants that permit the LFA 1/ICAM 1 complex to resist large pulling forces from the determinants that regulate the binding affinity of the complex.

Aim 4: Determine the contribution of LFA 1 clustering toward enhanced lymphocyte adhesion following cell activation.

Enhanced lymphocyte adhesion involves both changes in the affinity (i.e., the interaction between two molecules) and avidity (i.e., the number of interactions) states of LFA 1. We will use a J2.7 cell line that expresses a form of LFA-1 that is locked in the low affinity state to determine the relative contribution of avidity modulation in cell adhesion following cell activation. Moreover, we postulate that an important underlying mechanism for the avidity modulation of LFA 1 is the lateral redistribution of LFA 1 into clusters. To test this hypothesis, we will determine the effects of an induced clustering of LFA 1 on lymphocyte adhesion. Strategies for induction of LFA-1 clusters include (i) the stimulation of cells with phorbol ester and (ii) the crosslinking of LFA-1 with monoclonal antibody.

Aim 5: Determine if ICAM 1 dimerization augments the binding force of the LFA 1/ICAM 1 interaction.

ICAM 1 is expressed predominately as a dimer on the surface of cells. The working hypothesis for this aim is that dimerization of ICAM 1 may lend to “cooperative” molecular adhesion, whereby 2 ICAM 1 molecules act together as a single cooperative adhesion unit. Consequently, ICAM 1 dimers are predicted to have higher binding forces than ICAM 1 monomers acting singularly. To test this hypothesis, we have generated CHO cell lines that express either the native ICAM 1 dimer or the engineered ICAM 1 DAF monomer. We propose to carry out AFM force measurements to determine the dynamic strength of the LFA 1/native ICAM 1dimer complexes and of LFA 1 bound to the GPI-anchored monomeric ICAM 1. In parallel studies, we will also carry out measurements to determine if purified ICAM-1 dimers immobilized on a solid support are capable of mediating cooperative adhesion. Moreover, we have also devised experiments to determine if receptor cooperativity is dependent on the separation distance between ICAM 1 monomers.

The research will employ a state-of-the-art AFM to acquire direct force measurements of the interaction between LFA 1 and ICAM 1. Our preliminary studies demonstrated that this novel approach offers the sensitivity and temporal resolution to detect the breakage of individual linkages in a multicomponent system. The P.I. has carried out pioneering work in this area of research and is well qualified to carry out the proposed studies. The long-term benefits to be gained from the proposed research will be a better understanding of the mechanisms of cell adhesion in the immune system and consequently the development of better strategies for its modulation.