FIG. 4.

The excessive responses of splenocyte and splenic T-cells in vitamin C-insufficient Gulo(−/−) mice in response to Con A. Splenocytes or splenic T-cells were isolated from WT [Gulo(+/+)], vitamin C-insufficient [Gulo(−/−)], and vitamin C-sufficient [Gulo(−/−)+VC] mice as described in the Materials and Methods section and stimulated with Con A (5 μg/1×10⁶/ml) in vitro. (A, B) The excessive production of cytokines from splenocytes and splenic T-cells of vitamin C-insufficient Gulo(−/−) mice in response to Con A. After 48 h in vitro stimulation of Con A, the amounts of cytokines in the supernatants were determined by CBA or ELISA [n=10, *p<0.05 or **p<0.01 or ***p<0.001 vs. Gulo(−/−), ^†p<0.05 or ^†††p<0.001 vs. Gulo(−/−)+VC]. (C, D) The excessive proliferation of splenocytes and splenic T-cells from vitamin C-insufficient Gulo(−/−) mice in response to Con A. After 90 h in vitro stimulation of Con A, [³H]-thymidine incorporation was measured with a liquid scintillation counter as described in the Materials and Methods section [n=10, **p<0.01 vs. Gulo(−/−), ^†p<0.05 vs. Gulo(−/−)+VC]. (E) The frequency of the activated T-cells in splenocytes. After 48 h in vitro stimulation of Con A, the splenocytes were subjected to flow cytometry analysis of CD3⁺ CD25⁺ cells as described in the Materials and Methods section (n=5).