PECs (1 × 106/well), pretreated with
or without TGFβ2 at 5 ng/ml in 24-well plates, were cultured in 0.5
ml serum-free medium. After overnight culture, the plates were washed
three times with culture medium to remove TGFβ2 and nonadherent
cells. DO11.10 T cells (3 × 105) were added
in 24-well plates containing TGFβ2-treated, or -untreated PECs and
100 μg/ml OVA. After 24 hours, nonadherent cells (>98% T cells)
were collected and cultured (1 × 106/well)
in serum-free medium in 24-well plates for an additional 24 hours.
Supernatants were collected, and biologically active TGFβ was
measured using Mv1Lu cells (ATCC, Rockville, MD). To detect mature
TGFβ, the supernatants were diluted 1:4 with Eagle’s minimum
essential medium (EMEM) (Biowhitaker), which consisted of 2 mM l-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids,
1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin,
and 0.5% fetal calf serum. Diluted supernatants (100 μl) were added
to 96-well flat-bottomed plates. To measure total TGF-β, supernatants
were pretreated with 1N HCl (1:10) for 1 hour, then neutralized with a
mixture of 1N NaOH:1 M HEPES (1:5). These acidified supernatants were
diluted 1:10 with complete EMEM containing 0.5% fetal calf serum, then
100 μl was added to 96-well flat-bottomed plates. Mv1Lu cells (1 × 105 cells/100 μl) were added to each well
and cultured for 24 hours at 37°C in 5% CO2.
Cultures were pulsed with 1 μCi [3H]thymidine
6 hours before termination and harvested onto glass filters using an
automated cell harvester. Radioactivity was assessed as described
above. Half-maximal inhibition was determined by polynomial regression
on log–log transformation of standard curves and experimental samples.
The results were expressed as picograms per milliliter.