Immunohistochemistry. Oxazolone (Ox)-induced CHS was performed by using 2.0% and 1.0% Ox in ethanol for sensitization and challenge, respectively (1). Twenty four hours after Ox or vehicle challenge on day 5 after sensitization, ear pinnae were embedded in OCT compound and frozen in liquid N2, then 4-m m-thick serial sections were air-dried overnight, fixed in acetone (10 min at 4°C), rehydrated in PBS, and then incubated with 10% normal goat serum (10 min at room temperature). To detect mast cells, sections were incubated with FITC-avidin for 10 min at room temperature. The sections were then washed with PBS for 30 min, and endogenous biotin was blocked. To detect T cells, the sections were incubated with anti-CD3 mAb (145-2C11, BD Pharmingen) at 4°C overnight and washed with PBS for 30 min. The sections were incubated with biotinylated anti-hamster Ig (BD Pharmingen) at room temperature for 1 h, washed with PBS for 30 min, and then incubated with rhodamine-avidin at room temperature for 1 h.

Mice. C57BL/6-OTII transgenic mice were provided by R. Schwartz (National Institute of Allergy and Infectious Diseases, National Institutes of Health) through Taconic Farms (Germantown, NY).

Preparation of T Cell Subsets. A single-cell suspension of spleen cells was prepared, and red blood cells were lysed in RBC lysing buffer (Sigma). For CD3⁺ T cell purification, spleen cells were incubated with biotinylated anti-mouse B220 (RA3-6B2), Gr-1 (RB6-8C5), CD11b (M1/70), CD11c (N418), CD49b (DX5), Ter119 (Ter119), and c-kit/CD117 (2B8) for 20 min at 4°C. All antibodies were from eBioscience. For purification of CD4⁺ or CD8⁺ T cells, spleen cells were incubated with biotinylated antibody as described above plus biotinylated anti-mouse CD8 (53-6.7, BD Pharmingen) or CD4 (RM4-5, BD Pharmingen), respectively. The cells were then washed and incubated with Streptavidin beads (Miltenyi Biotec) for 20 min at 4°C, and washed again and passed through a magnetic cell-sorting column (Miltenyi Biotec), resulting in fractions that contained >95% CD3⁺, CD4⁺, or CD8⁺ T cells, respectively (Fig. 12A). For CD4⁺CD62L^- T cell purification, CD4⁺ T cells were incubated with biotinylated anti-mouse CD62L (Mel-14, eBioscience) for 20 min at 4°C. After washing and Streptavidin bead incubation, CD4⁺CD62L^- T cells were separated by magnetic cell-sorting columns (>90% CD4⁺CD62L^- T cells (Fig. 12B). For g d TCR⁺ T cell purification, CD3⁺ T cells were incubated with FITC anti-mouse g d TCR (GL3, BD Pharmingen) for 20 min at 4°C, washed, incubated with anti-FITC-beads (Miltenyi Biotec) for 20 min at 4°C, washed again and then passed through magnetic cell-sorting columns to collect column-bound g d TCR⁺ T cells (>80% g d TCR⁺ T cells) (Fig. 12C).

Preparation of Th1/Tc1 and Th2/Tc2 Cells. To obtain Th1 or Tc1 cells (Fig. 12 D and E), CD4⁺ or CD8⁺ T cells were cultured with 1 m g/ml plate-coated anti-mouse CD3 (145-2C11, BD Pharmingen) in the presence of 10 ng/ml rmIL-2 (R&D Systems, Minneapolis), 1 ng/ml rmIL-12 (R&D Systems), and 10 m g/ml anti-mouse IL-4 (11B11, BD Pharmingen) for 4 days. To obtain Th2 or Tc2 cells (Fig. 12 D and E), CD4⁺ or CD8⁺ T cells were cultured with 1 m g/ml plate-coated anti-mouse CD3 in the presence of 10 ng/ml rmIL-2 (R&D Systems), 20 ng/ml rmIL-4 (PeproTech), and 10 m g/ml anti-mouse IFN-g (XMG1.2, BD Pharmingen) for 5 days. Then, IL-4⁺ or IFN-g ⁺ cells are purified by using a cell enrichment and detection kit (Miltenyi Biotech).

ELISAs for Cytokine Measurements. Cytokine levels in culture supernatants were measured by using mouse IL-4, IL-17, and IFN-[BD OptEIA ELISA Sets (BD Pharmingen)].

I-A^b Expression by Flow Cytometry. For I-A^b expression on BMCMCs alone, BMCMCs were sensitized with or without 10 m g/ml of the H1-e -26 monoclonal IgE anti-DNP Ab at 37°C overnight. After washing the cells, BMCMCs were cultured in the absence or presence of 10 ng/ml DNP-HSA for 6 h. In T cell-BMCMC cocultures, CD3⁺ T cells were cultured with IgE-sensitized BMCMCs in the absence or presence of anti-CD3 mAb or DNP-HSA for 72 h as described in Fig. 1A. The cells were then harvested and stained with PE-anti-mouse I-A^b mAb (AF6-120.1, BD Pharmingen) or PE-mouse IgG2a for isotype-matched control Ab after FcR blocking. For I-A^b expression on dendritic cells, single-cell suspensions of spleen cells from C57BL/6 mice were prepared and then spleen cells were incubated with FITC-anti-mouse CD11c (HL3, BD Pharmingen) and PE-anti-mouse I-A^b mAbs after FcR blocking. Expression of cell surface markers was analyzed on a FACS Calibur (Beckton Dickinson) by using CELLQUEST software (Becton Dickinson).

OVA-Specific T Cell Proliferation. Whole spleen cells or splenic CD4⁺ T cells were prepared from spleens of OTII transgenic mice as described above. OTII spleen cells, or OTII CD4⁺ T cells together with mitomycin C-treated, non-IgE-sensitized BMCMCs, were cultured in the absence or presence of OVA323-339 peptides. Proliferation was assessed by pulsing with 0.25 m Ci [³H]thymidine (Amersham Pharmacia) for 6 h, harvesting the cells using Harvester 96 Mach IIIM (Tomtec) and measuring incorporated [³H]thymidine using the Micro Beta system (Pharmacia Biotech).

1. Bryce, P. J., Miller, M. L., Miyajima, I., Tsai, M., Galli, S. J. & Oettgen, H. C. (2004) Immunity 20, 381-392.