MATERIALS AND METHODS

Antibodies and reagents.
The following antibodies used for intracellular or surface stains were obtained from eBioscience: anti–IL-2, anti–IFN-γ, anti-TNF, anti–granzyme B, anti-CD8, anti-CD25, and anti-CD44. Anti-CD69 was purchased from BD. For ChIP experiments, the anti-Eomes antibody was obtained from Abcam and the anti-Runx3 antibody was produced by the Groner laboratory. The following antibodies were used for immunoblotting: antiperforin (Abcam), anti-Eomes (Abcam), and anti–Pol-II (Santa Cruz Biotechnology, Inc.). The T-bet antibody was provided by L. Glimcher (Harvard School of Public Health, Boston, MA).
The following reagents were used for the experiments presented in this report: Annexin V–FITC Apoptosis Detection Kit (BD), CD8 Negative Isolation Kit (Invitrogen), CD8 MicroBeads (Miltenyi Biotec), and SYBR Green PCR Core Reagents (Applied Biosystems). The Gp33 peptide (KAVYNFATC) was synthesized by the Tufts University Core Facility, and 10 mM of stock solutions was prepared in DMSO.

Isolation and culture of primary CD8+ T cells.
CD8+ T cells from 4–8-wk-old Tcra−/− × P14 TCR transgenic (Taconic), C57BL/6J WT, or Tbx21−/− (The Jackson Laboratory) mice were purified (>95% purity) by negative selection (Invitrogen) from pooled spleen and lymph node cells. CD8+ T cells from Runx3−/− mice on the ICR background were purified by positive selection (Miltenyi Biotec). All mice were maintained in specific pathogen-free barrier facilities and used according to protocols approved by the Immune Disease Institute and the Harvard Medical School Animal Care and Use Committees. For stimulation, purified CD8+ T cells were cultured at 106 cells/ml (10 ml) in T25 flasks coated with 1 μg/ml each of anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) by pretreatment with 300 μg/ml goat anti–hamster IgG. After 48 h, cells were removed from the TCR stimulation and recultured at a concentration of 5 × 105 cells/ml in media supplemented with 100 U/ml rhIL-2. Every 24 h, viable cells were counted and readjusted to 5 × 105 cells/ml with fresh media containing the corresponding amount of rhIL-2.

Isolation of CD8+ T cells from Runx3−/− mice.
Runx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13). We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4− SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads. This yielded a Runx3 KO SP “enriched” population that contained 75% CD8+CD4− cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1). The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2. As previously reported, TCR-induced proliferation of Runx3−/− CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13). However, the Runx3−/− cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1). As expected from their ability to up-regulate CD25, Runx3−/− CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1). Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3−/− CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not. The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs. 3 and 4 are from total Runx3 KO CD8 cells.

FACS-based cytotoxicity assay.
To measure cytotoxicity, EL4 thymoma target cells were loaded with 0 or 1 μM Gp33 peptide for 2 h before a 2-h coincubation with P14 CD8+ T cells at the effector-to-target ratios indicated in the figures in 96-well round-bottom plates. After the coincubation period, cells were stained with Annexin V–FITC and anti-CD8–allophycocyanin. Data analysis was performed with FlowJo software (Tree Star, Inc.); EL4 target cells (CD8-negative events) were gated, and the percentage of Annexin V+ target cells was determined.

Cytokine and surface marker staining.
To assess cytokine production, cells were restimulated with 10 nM PMA + 1 μM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28). To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1× PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD).

Retroviral transduction of primary CD8+ T cells.
For transduction experiments, viral supernatants were generated by calcium phosphate transfection of Phoenix cells and concentration by overnight centrifugation at 6,000 g. At ∼42 h after the initial TCR activation of 106 CD8+ T cells per well in 12-well plates, the culture media was removed and replaced with complete media supplemented with 8 μg/ml polybrene containing fresh plus concentrated virus. The plates were centrifuged at 700 g for 1 h at RT before returning to 37°C for an additional 5 h. Retroviral constructs for Eomes-VP16 and the MIG control empty vector were a gift from S.L. Reiner (University of Pennsylvania, Philadelphia, PA) (8).

ChIP and real-time PCR analysis.
20 × 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT. Fixation was stopped with 120 mM glycine on ice for 5 min. Fixed cells were washed 2× with cold PBS, 1× with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1× with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). After washes, cell pellets were resuspended at 40 × 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5–1-kb DNA fragments. After preclearing the sheared chromatin with protein A–sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 × 106 cell equivalents: 2.5 μg anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4°C; protein A–sepharose beads were added for the last 3 h of the incubation period. Beads were washed 2× with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1× with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1× with TE buffer. After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3). Both input and ChIP chromatin were then treated with RNase A (5 μg total) for 1 h at 37°C, followed by the addition of proteinase K (100 μg total) and overnight incubation at 65°C to reverse cross-linking. DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-μl volume. For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 μl ChIP DNA was used per PCR reaction (performed in duplicates). Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences. Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets. The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the ChIP.

Northern and Western blot analyses.
RNA isolation and Northern blot analysis was performed as previously described (29). In brief, 10 μg of total RNA was loaded per lane and transferred to positively charged nylon membranes (Hybond-N+; GE Healthcare), which was confirmed by ethidium bromide staining of ribosomal RNA species on the membrane. Membranes were hybridized with 1 ng/ml α-[32P]dCTP–labeled trichloroacetic acid precipitable probe in ExpressHyb hybridization buffer (Clontech Laboratories, Inc.). All cDNA probes were confirmed to have the appropriate single-copy specificity under these conditions using genomic Southern blot analysis. Band intensities were acquired by phosphorimaging analysis.
For Western analysis, whole-cell protein lysates were obtained from CD8+ T cells at the time points indicated in the figures during clonal expansion in 100 U/ml IL-2 with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40) by resuspending samples in 10 μl per 106 cells and incubating on ice for 30 min in the presence of protease inhibitors. Immunoblot analysis was performed with the antibodies indicated in the figures after SDS-PAGE (10–30 μg of total protein was loaded per well). Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of RNA Pol-II detected in each lane.

Online supplemental material.
Fig. S1 shows the characterization of peripheral CD8+ T cells from Runx3−/− mice. Fig. S2 shows effector protein expression by Runx3 WT and KO cells at day 4 of in vitro culture. Primer sequences used for ChIP experiments are shown in Table S1. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081242/DC1.