Engineering of synthetic cellular microenvironments: implications for immunity.

In this article, we discuss novel synthetic approaches for studying the interactions of cells with their microenvironment. Notably, critical cellular processes such as growth, differentiation, migration, and fate determination, are tightly regulated by interactions with neighboring cells, and the surrounding extracellular matrix. Given the huge complexity of natural cellular environments, and their rich molecular and physical diversity, the mission of understanding "environmental signaling" at a molecular-mechanistic level appears to be extremely challenging. To meet these challenges, attempts have been made in recent years to design synthetic matrices with defined chemical and physical properties, which, artificial though they may be, could reveal basic "design principles" underlying the physiological processes. Here, we summarize recent developments in the characterization of the chemical and physical properties of cell sensing and adhesion, as well as the design and use of engineered, micro- to nanoscale patterned and confined environments, for systematic, comprehensive modulation of the cells' environment. The power of these biomimetic surfaces to highlight environmental signaling events in cells, and in immune cells in particular, will be discussed.

Fine tuning and efficient T cell activation with stimulatory aCD3 nanoarrays.

Anti-CD3 (aCD3) nanoarrays fabricated by self-assembled nanopatterning combined with site-directed protein immobilization techniques represent a novel T cell stimulatory platform that allows tight control over ligand orientation and surface density. Here, we show that activation of primary human CD4+ T cells, defined by CD69 upregulation, IL-2 production and cell proliferation, correlates with aCD3 density on nanoarrays. Immobilization of aCD3 through nanopatterning had two effects: cell activation was significantly higher on these surfaces than on aCD3-coated plastics and allowed unprecedented fine-tuning of T cell response.

T cell activation is determined by the number of presented antigens.

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per µm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

Impact of local versus global ligand density on cellular adhesion.

α(v)ß(3) integrin-mediated cell adhesion is crucially influenced by how far ligands are spaced apart. To evaluate the impact of local ligand density versus global ligand density of a given surface, we used synthetic micronanostructured cell environments with user-defined ligand spacing and patterns to investigate cellular adhesion. The development of stable focal adhesions, their number, and size as well as the cellular adhesion strength proved to be influenced by local more than global ligand density.

Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy.

During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen-presenting cells (APCs). This recognition results in the formation of a so-called immune synapse (IS) at the T-cell/APC interface, which is crucial for T-cell activation. The molecular composition of the IS has been extensively studied, but little is known about the biophysics and interaction forces between T cells and APCs. Here, we report the measurement of interaction forces between T cells and APCs employing atomic force microscopy (AFM). For these investigations, specific T cells were selected that recognize an antigenic peptide presented by MHC-class II molecules on APCs. Dynamic analysis of T-cell/APC interaction by AFM revealed that in the presence of antigen interaction forces increased from 1 to 2 nN at early time-points to a maximum of approximately 14 nN after 30 min and decreased again after 60 min. These data correlate with the kinetics of synapse formation that also reached a maximum after 30 min, as determined by high-throughput multispectral imaging flow cytometry. Because the integrin lymphocyte function antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule-1 (ICAM-1) are prominent members of a mature IS, the effect of a small molecular inhibitor for LFA-1, BIRT377, was investigated. BIRT377 almost completely abolish the interaction forces, emphasizing the importance of LFA-1/ICAM-1-interactions for firm T-cell/APC adhesion. In conclusion, using biophysical measurements, this study provides precise values for the interaction forces between T cells and APCs and demonstrates that these forces develop over time and are highest when synapse formation is maximal.