T-Cell Artificial Focal Triggering Tools: Linking Surface Interactions with Cell Response

Benoît Carpentier,Nelly Henry,Claire Hivroz,Paolo Pierobon,Derya Unutmaz

doi:10.1371/journal.pone.0004784

Abstract

T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy.

Highlights

T-cell activation plays a central role in the mammalian immune response [1]
T cells are activated via engagement of Tcell receptors (TCRs) with antigenic peptides presented in the cleft of major histocompatibility complex (MHC) molecules at the surface of antigen-presenting cells (APCs) [4]
Because a biotin-antibody chemical link was made up of a dozen sp3 carbons, we assumed that monoclonal antibodies (mAb) molecules which bound to the particle via the biotin anchor were free to rotate so as to find their target on the cell surface

Summary

Introduction

T-cell activation plays a central role in the mammalian immune response [1]. It is the mainspring of several immunotherapeutic strategies [2,3]. In order to take this into account, strategies consisting of replacing one of the cells in the interacting pair by a synthetic surface bearing appropriate Tcell ligands have been developed using either polymer microparticles [12,13,14,15] or planar surfaces made up of supported lipid bilayers or monolayers on solid substrates [16,17] They constitute rather crude cell models, solid microspheres represent interesting investigative tools, since they enable exact specification of the nature and density of the ligand presented to the cell surface. As used by Wei et al [12], consists of using micromanipulation techniques to present the microsphere to the cell surface prior to imaging the cell response through intracellular cell calcium This enables investigating the process at the single cell level and provides important qualitative information; it requires examining cells one by one, which is very timeconsuming and limits the sample size, whereas variation between cells may be high. It cannot be implemented for examining several receptor classes or combinations, which is necessary for complex processes like T-cell activation

Methods

Results

Discussion

Conclusion