Inducing local T cell apoptosis with anti-Fas-functionalized polymeric coatings fabricated via surface-initiated photopolymerizations

Abstract

Cell encapsulation has long been investigated as a means to achieve transplant immunoprotection as it creates a physical barrier between allograft tissue and host immune cells. Encapsulation with passive barrier materials alone, however, is generally insufficient to protect donor tissue from rejection, because small cytotoxic molecules produced by activated T cells can diffuse readily into the capsule and mediate allograft death. As a means to provide bioactive protection for polymeric encapsulation devices, we investigated a functionalized polymeric coating that mimics a natural T cell regulation pathway. T cells are regulated in vivo via Fas, a well-known 'death receptor,' whereby effector cells express Fas ligand and elicit T cell apoptosis upon binding the Fas receptor on a T cell surface. Anti-Fas antibodies are capable of replicating this effect and induce T cell apoptosis in solution. Here, an iniferter-based living radical polymerization was utilized to fabricate surface-anchored polymer chains containing poly(ethylene glycol) with covalently incorporated pendant anti-Fas antibody. Using this reaction mechanism, we demonstrate fabrication conditions that yield surface densities in excess of 1.5 ng/cm(2) of incorporated therapeutic, as detected by ELISA. Additionally, we show that coatings containing anti-Fas antibody induced significant T cell apoptosis, 21+/-2% of cells, after 24h. Finally, the incorporation of a T cell adhesion ligand, intracellular adhesion molecule-1, along with anti-Fas antibody, yielded even higher levels of apoptosis, 34+/-1% of T cells, compared to either signal alone.

Fig. 1

Schematic illustrating surface-initiated polymerization of acrylated proteins. ACRYL-protein is co-photopolymerized with ACRYL-PEG atop a polymeric substrate containing a DTC photoiniferter. Polymer chains consisting of polyacrylate backbones with pendant proteins are formed on the surface, and the surface modification is proportional to UV exposure time.

Fig. 2

The influence of covalent acrylation on the bioactivity of soluble ACRYL-DX2. ACRYL-PEG-NHS was reacted with DX2 in the molar ratios shown on the x-axis. As the reaction stoichiometry was increased, the efficacy with which soluble ACRYL-DX2 induced apoptosis was reduced.

Fig. 3

Controlled surface-initiated photopolymerization of ACRYL-IgG. (A) Dry graft height, determined by profilometry, increases as a function of surface-initiated polymerization time. (B) Surface density of detectable ACRYL-IgG increases and then decreases as a function of polymerization time, as determined by modified ELISA. A region of high detectable ACRYL-IgG exists between 120 – 180 s fabrication times. (C) Fluorescein-tagged ACRYL-IgG from goat (F-ACRYL-IgG) was photografted for 150 and 500 s. Dashed boxes represent a cross-sectional view of the full thickness of each coating. Fluorescein is visible throughout the thickness of both samples. Scale bars = 50 μm. (D) F-ACRYL-IgG Grafts were stained with rhodamine-tagged donkey anti-goat IgG (R-IgG) to label accessible F-ACRYL-IgG. Strong full-thickness R-IgG staining is visible at 150 s but only surface staining at 500 s. Scale bars = 50 μm. (E) Surface density of detectable ACRYL-IgG decreased as a function of TED iniferter concentration in the substrate.

Fig. 4

Grafted ACRYL-DX2 induces T cell apoptosis. (A) ACRYL-DX2 was incorporated in 6 mm diameter grafts and incubated with HRP-conjugated GAM IgG (left) or soluble Fas receptor, goat anti-Fas IgG, and HRP-conguated DAG IgG (right). Both (left) and (right) were stained with Vector VIP to stain HRP. Vector VIP staining indicates (left) high ACRYL-DX2 surface density and (right) ACRYL-DX2 retains the ability to bind the Fas receptor. (B & C) Representative brightfield (right) and 480 nm fluorescent (left) images of Jurkat T cells seeded for 24 hrs on grafted (B) control and (C) ACRYL-DX2 surfaces for 24 hrs followed by staining with fluorescein-conjugated Annexin V. Apoptotic T cells are visible at 480 nm. Scale bars = 100 μm (D) Jurkat and Fas-insensitive I9.2 T cells were seeded on grafted surfaces for 24 hours and assayed for apoptosis. A statistically significant increase in apoptosis was observed for Jurkat T cells incubated on ACRYL-DX2 surfaces. Asterisks indicates a statistically significant difference (p<0.05) from all other values.

Fig. 5

ACRYL-ICAM-1 improves the efficacy of grafted ACRYL-DX2. (A) Grafted ACRYL-ICAM-1, when incorporated with ACRYL-DX2, increases the percentage of Jurkat T cells signaled to undergo apoptosis after 24 hrs. (B) Metabolic activity studies of Jurkat T cells seeded on control or dually-functionalized grafted surfaces for 24 hrs. Jurkat T cells show an over 50% reduction in metabolic activity when cultured on ACRYL-DX2 / ACRYL-ICAM-1 grafts. Asterisks indicates a statistically significant difference (p<0.05) from all other values.