Similar to IL-2 secretion (Fig. cells cultured around the stiffer substrates also demonstrate a trend (non-significant) towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Na?ve CD4+ T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN- producing TH1-like cells. These results reveal that this rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation and TH differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands. test for paired data, Wilcoxon Rank Sum or a one-way analysis of variance (ANOVA) were performed using GraphPad Prism version 4.0a (GraphPad Software Inc.). A p-value of 0.05 was considered statistically significant. Results PDMS as a substrate with controllable rigidity for T cell activation and culture PDMS, a biocompatible organosilicon polymer commonly used as a lubricant, anti-caking agent in foods and anti-bloating agent was selected as a substrate for antibody immobilization. Following crosslinking of the base polymer, PDMS forms an elastomeric material with a highly hydrophobic surface [21]. Proteins, including antibody, passively L-ANAP adsorb to this hydrophobic surface. Alteration of the crosslinking-agent-to-base-polymer stoichiometry in the commonly used Sylgard 184 preparation of PDMS provides a simple method for varying the elastic modulus of PDMS from a Youngs modulus of 2.3MPa (stiff) to a range of 50-100 kPa (soft) (Fig. 1A). Prepared this way, this material has been used to study the effects of substrate rigidity on fibroblast focal adhesion formation [4]. Adsorption of anti-CD3 (OKT3) and anti-CD28 (clone 9.3) antibodies to the surface of PDMS provides a system for activation of T cells on substrates with varying elastic modulus, analogous to standard immobilization on more rigid polystyrene tissue culture plastic or glass. Quantitative measurement of enzymatically-coupled primary capture antibody (Fig. 1B) as well as fluorescently-labeled OKT3 and clone 9.3 (data not shown) demonstrate that the amount of antibody adsorbed on PDMS surfaces with varying elastic modulus is equivalent despite changes in the ratio of base polymer to crosslinking agent. Both OKT3 and clone 9.3 also demonstrated stable binding over the course of 48 hours with 20% loss of antibody at 37C in complete culture medium independent of the crosslinker ratio (Fig. 1C). Clone Rabbit Polyclonal to HARS 9.3 appeared to demonstrate a slightly more rapid loss from stiff surfaces compared soft surfaces; however, the quantity of bound clone 9.3 was not significantly different between the PDMS surfaces at 48 hours, after which T cells are typically transfer to uncoated culture vessels for log-phase ex vivo expansion using planar activating substrates. Open in a separate window Physique 1 A T cell culture surface with controlled elastic modulus can be generated using L-ANAP variably L-ANAP cross-linked PDMS(A) The elastic modulus of PDMS was measured as described in the Materials and Methods. Horizontal bars represent the mean of four impartial batches of PDMS. (B) PDMS surfaces were coated with the indicated concentration of biotinylated goat-anti-mouse IgG. Adsorbed antibody was detected by incubation with horseradish peroxidase conjugated to streptavidin and TMB followed by measurement of the optical density (OD) at 450 nm. Data presented is representative of two impartial experiments. Symbols indicated L-ANAP replicate wells performed within the experiment. (C) Fluorescently-conjugated antibodies against CD3 (OKT3) and CD28 (9.3) were simultaneously applied to PDMS surfaces pre-coated with goat-anti-mouse IgG at L-ANAP 5 mg/mL followed by washing and blocking. The fluorescent signal.
