Arrays for T Cell Adhesion and Surface Phenotype Analysis. Biotinylated MHC-peptide complexes (³ 0.5 mg/ml) were mixed in a 4:1 molar ratio with unlabeled SA in PBS (pH 7.4) and allowed to form tetramers at ambient temperature. The mixture was diluted in PBS to give a final concentration of 50 m g/ml MHC complex (≈14 m g/ml SA). Adhesion/costimulatory molecules anti-CD11a, anti-CD2, and/or anti-CD28 antibodies were added to a final concentration of 5 m g/ml, and the mixture was spotted at room temperature onto microscope slides by using a manual pipettor [Lambda 0.1-2 m l single-channel pipettor (Corning)], giving 0.5 m l spots spaced 2 mm apart on centers; by using a semiautomatic solid-pin contact arrayer (Xenopore manual microarrayer with 500-m m-diameter solid pins), giving spots 0.75-1.13 mm apart on centers; or by using an automatic, noncontact array printer [Cartesian synQUAD technology (Genomic Solutions)], giving 50- to 75-nl spots spaced 1.0-1.5 mm apart on centers. The spots were contained within a barrier that could contain a single pool of media: either a barrier drawn with hydrophobic ink (Ted Pella, Inc.), a removable rubber gasket (Molecular Probes), or a removable plastic well [LabTek chamber slide (Nalge)]. The protein solution was allowed to dry completely at room temperature, and the resulting array was stored at 4°C in a dessicator until use.

Arrays for Cytokine Capture. Cytokine-capture monoclonal antibodies (BD Pharmingen) were diluted in PBS (pH 7.4) to a final concentration of 40 m g/ml. The antibody solution was spotted onto microscope slides at room temperature, as described, and allowed to dry. The antibody solution was spotted again on the dry spots and again allowed to dry until four layers of antibody solution had been spotted. Solutions of biotinylated MHC–peptide tetramers with or without costimulatory/adhesion antibodies prepared as above were spotted at room temperature on top of the dried cytokine-capture spots, placing different MHC–peptide complexes in different locations on the slide. The MHC-containing solution was spotted only one time. The array was allowed to dry completely at room temperature and was stored at 4°C in a dessicator until use.

T Cell Incubation. Dried arrays were rehydrated and blocked by addition of RPMI medium 1640-based T cell medium containing 10% FCS over the entire slide within the barrier and incubated for 30 min at room temperature. After blocking, the medium was removed, and cell samples (T cell clones, lines, or peripheral blood mononuclear cells) were added over the entire slide in fresh T cell medium. The number of T cells added varied in different experiments, depending on the total wetted surface area of the array and the frequency of responding cells in the sample. Clear responses were observable T cell clones at 10⁴-10⁶ cells over an entire slide. Higher densities of cells were used for detection of low-frequency responses. Enough medium was added to have ≥1 mm thickness of liquid over the entire array. The arrays with T cells were incubated at 37°C, in 5% CO₂ for varying amounts of time.

T Cell-Adhesion and Surface-Phenotype Analysis. Specific adhesion was seen within 30 min of T cell addition to the blocked arrays, but longer incubations were used for development of certain surface phenotypes, including TCR down-regulation and CD69 up-regulation. After incubation, the arrays were chilled to 4°C, and then Hoechst nuclear stain (Molecular Probes) and fluorescent antibodies to cell-surface proteins (BD Pharmingen) were added to the media on the slide and incubated at 4°C for 40 min. The medium was then aspirated off, and a solution of 1% paraformaldehyde in 1× PBS was added onto the slide for a 5-min incubation at room temperature. The 1% paraformaldehyde solution was then removed, and the slide was rinsed twice with 1× PBS and then dehydrated by rinsing with 70% ethanol. A glass coverslip was applied with VECTASHIELD (Vector Laboratories), and cell adhesion and surface phenotype were observed in different array areas by using a Nikon Eclipse E800 fluorescence microscope.

Cytokine-Capture Analysis. IL-2, IFN-g , and granzyme were detected 4-16 h after T cell addition to the blocked arrays, with an optimal incubation time of 6-8 h, but other cytokine responses may have different optimal detection times. After incubation, the media and T cells were removed, and a series of washes were performed: two 5-min incubations in ddH₂O at room temperature, followed by three rinses with Tris-buffered saline (pH 7.6) containing 0.1% Tween 20.

Fluorescent detection of captured cytokine. Biotinylated monoclonal cytokine-detection antibody at 0.5 mg/ml [paired to cytokine-capture antibody (BD Pharmingen)] was preincubated at room temperature for 30 min at a 4:1 volume ratio with fluorescently labeled SA at 2 mg/ml, where the fluorescent probes were chosen for detection in an Affymetrix 428 array scanner [CY3, CY5, Alexa Fluor 555, or Alexa Fluor 647 (Molecular Probes)]. After this preincubation, the mixture was diluted into Tris-buffered saline (pH 7.6) containing 0.1% Tween 20, 0.3% BSA, and 100 m M free biotin. The final concentrations of both the antibody and fluorescent SA are 2 m g/ml each. Any additional complementary fluorescently labeled antibodies (e.g., conformationally specific antibodies to detect native MHC–peptide-complex immobilization) were added into this staining solution at 0.5-1.0 m g/ml. The staining solution was incubated on the array at room temperature in the dark for 1.5 h. The staining solution was removed, and the array was rinsed three times with Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and was then rinsed two more times with ddH₂0. The rinsed array was air-dried and could be stored dry in the dark at room temperature until detected by using an Affymetrix 428 array scanner.

Precipitating substrate detection of captured cytokine. Biotinylated monoclonal cytokine-detection antibody at 0.5 mg/ml [paired to cytokine-capture antibody (BD Pharmingen)] was preincubated at room temperature for 30 min at a 4:1 volume ratio with SA-horseradish peroxidase (Sigma) at 2 mg/ml. After this preincubation, the mixture was diluted into Tris-buffered saline (pH 7.6) containing 0.1% Tween 20, 0.3% BSA, and 100 m M free biotin. The final concentrations of both the antibody and SA-horseradish peroxidase are 2 m g/ml each. The detection solution was incubated on the array at room temperature for 1.5 h. Fifteen minutes before the end of the incubation with detection solution, the 3-amino-9-ethylcarbazole substrate solution was prepared by adding 20 m l of substrate (BD Pharmingen) per ml of PBS and mixing well. Then, the detection solution was removed, and the array was rinsed three times with Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and then two times with 1× PBS (pH 7.4) making sure that no azide was present. The rinsed array was coated with the 3-amino-9-ethylcarbazole substrate solution and allowed to react in the dark at room temperature for 5-60 min, while periodically visually monitoring progress. The reaction was stopped by removing the substrate solution and rinsing twice with ddH₂O, and the array was allowed to dry completely. The spots were observed by eye and under a dissection microscope.