a) Fluorescent micrographs of a block of microwells containing PBMCs (labeled with Hoescht dye) and the matched region of the printed microarray of captured IFNγ. The array of microwells was coated with streptavidin (200 μg/mL), followed by biotinylated anti-CD3 (10 μg/mL) and anti-CD28 (1 μg/mL). The cells were incubated on the modified wells for 6 h at 37 °C prior to microengraving. The boxes (dashed lines) on the right hand image have been overlaid to clarify the regions of interest. The scale bar is 100 μm. b) A plot of the measured frequencies of IFNγ-secreting PBMCs after stimulation either directly on-chip for 6, 18, or 42 h (black) or in bulk in a 96-well microtiter plate (white) for 6, 18, or 42 h. After bulk stimulation, the cells were deposited into microwells for microengraving. The error bars represent the range in responses measured in three independent experiments. c) A plot of the frequencies of IFNγ-secreting (white) and IL-17-secreting (black) MOG-specific clones following on-chip stimulation for 6 h as a function of applied concentration of MOGp97-109-loaded HLA DRB1*0401 monomer (0, 2, 10, 30, 60 μg/mL).

Schematic illustration of surface modification for on-chip stimulation of antigen-specific T cells and subsequent detection of released cytokines by microengraving. Streptavidin is applied to oxidized PDMS, then exposed to biotinylated monomers of recombinant, peptide-loaded MHC Class II and biotinylated anti-CD28 (not shown for clarity). The modified surface is blocked with bovine serum albumin immediately prior to loading cells. Cells are then deposited into the microwells and incubated to allow activation. After a period of time (6-18 h), the array of cells is sealed against a glass slide bearing anti-cytokine antibodies and incubated for 2 h. After this process (microengraving), the array of cells is imaged to determine the number of cells per well, and the array of captured cytokines is labeled and imaged.

Plots of the frequencies of IFNγ-secreting (white) and IL-17 secreting (black) (a) MOG-specific and (b) HA-specific T cell clones following on-chip stimulation for 6 h. The stimulation conditions included streptavidin only (SA), MOGp97-109-loaded HLA DRB1*0401 monomer (10 μg/mL), HAp306-318-loaded HLA DRB1*0401 monomer (10 μg/mL), and anti-CD3 (OKT3; 10 μg/mL). All conditions, except streptavidin only, also included anti-CD28 (1 μg/mL) for co-stimulation. Both IFNγ (white bars) and IL-17 (black bars) were captured by microengraving after stimulation. The error bars indicate the range in frequencies measured from three independent experiments.