An Experimentally Determined State Diagram for Human CD4+ T Lymphocyte CXCR4-Stimulated Adhesion Under Shear Flow

Abstract

Introduction: The leukocyte adhesion cascade is important for the maintenance of homeostasis and the ability of immune cells to access sites of infection and inflammation. Despite much work identifying the molecular components of the cascade, and numerous simulations to predict the relationship between molecule density, identity, and adhesion, these relationships have not been measured experimentally.

Methods: Using surfaces functionalized with recombinant ICAM-1 and/or E-selectin along with immobilized SDF-1α, we used a flow chamber to measure rates of tethering, rolling and arrest of primary naïve human CD4⁺ T lymphocytes on different surface densities of ligand.

Results: Cells required a minimum level of ligand density to progress beyond tethering. E-selectin and ICAM-1 were found to have a synergistic relationship in promoting cell arrest. Surfaces with both ligands had the highest levels of arrest, while surfaces containing only E-selectin hindered the cell's ability to progress beyond rolling. In contrast, surfaces of ICAM-1 allowed only tethering or arrest. Cells maintained constant rolling velocity and time to stop over large variations in surface density and composition. In addition, surface densities of only O(10¹) sites/μm² allowed for rolling while surface densities of O(10²) sites/μm² promoted arrest, approximately equal to previously determined simulated values.

Conclusions: We have systematically and experimentally mapped out the state diagram of T-cell adhesion under flow, directly demonstrating the quantitative requirements for each dynamic state of adhesion, and showing how multiple adhesion molecules can act in synergy to secure arrest.

Keywords: CXCL12α; E-selectin; ICAM-1; SDF-1α; flow chamber; site density.

Figure 1

Schematic representation of T cell dynamic adhesion with a surface containing recombinant ICAM-1/Fc and E-selectin/Fc chimeras with SDF-1α.

Figure 2

State diagrams of the fraction of cells undergoing (a) tethering, (b) rolling, or (c) firm arrest. Red dots indicate experimentally tested points. All experiments were performed at a calculated wall shear rate of 100 s⁻¹.

Figure 3

Calculated surfaces showing the effect of ICAM-1 and E-selectin densities on (a) rolling velocity and (b) time to stop. Red dots indicate experimentally tested points.