FIG. 5.

The effect of ascorbic acid on T-cell maturation is cell intrinsic. OP9-DL1 stromal cells were cultured with 2×10³ FACS-sorted LSK-Thy-1.1-neg cells in the presence of 5 ng/ml each of IL-7 and Flt3L for 17 days, with passages every 3–4 days. On the 17th day of culture, nonadherent lymphoid cells were harvested by gentle rinsing of the OP9-DL1 monolayer and transferred to a new culture plate for 30 min to deplete residual OP9-DL1 cells by adherence. The lymphoid cells were then incubated at 37°C for 2 h with cytokines as above in the absence (Ly−) or presence (Ly+) of ascorbic acid (250 μM). In parallel, established monolayers of OP9-DL1 cells were also incubated in the absence (St−) or presence (St+) of the same concentration of ascorbic acid. After 2 h, all cell populations were washed, and cultures were established in which lymphocytes, stromal cells, both cell populations, or neither population was exposed to ascorbic acid. As a positive control, pAsc was added at a concentration of 250 μM to one set of cultures. After 6 days, quadruplicate cultures were harvested for cell counting and phenotypic analysis as indicated. Values represent mean±standard error. A two-tailed t-test was used for statistical analysis.