In a previous report, we showed that, on exposure in vitro to the immunizing peptide, in vivo primed CD8 IRBP-specific T cells proliferate marginally and produce limited amounts of proinflammatory cytokines, such as IL-2 and IFN-γ.
11 In the present study, we exposed purified CD4 and CD8 T cells from IRBP1-20-immunized mice to graded doses of immunizing antigen ranging from 0.1 to 10 μg/mL in the presence of syngeneic APCs (irradiated spleen cells); then, 48 hours later, the culture supernatants were assessed for cytokines, including IL-2 and IFN-γ, and the cells were cultured with exogenous IL-2 for a further 3 days and analyzed by PCR for Foxp3. As shown in
Figure 1 , CD8 cells exposed to a high dose of antigen (10 μg/mL) produced a modest level of cytokines
(Fig. 1A)and expressed limited levels of Foxp3
(Fig. 1B) . In contrast, after exposure to a low dose of immunizing antigen (0.1 μg/mL), these cells expressed much more Foxp3
(Fig. 1B)without producing a significant amount of cytokines
(Fig. 1A) , whereas the same T cells cultured with APCs alone in the absence of antigen did not express IFN-γ and expressed a very low level of Foxp3
(Figs. 1A 1B) . Thus, the expression of Foxp3 by CD8 IRBP-specific T cells correlated inversely with the degree of activation. To exclude the possibility that such cells were defective in cytokine-producing ability, we exposed the separated CD4 or CD8 T cells to 0.1 to 10 μg/mL of immunizing antigen or 1 to 1000 ng/mL of SEE in the presence of syngeneic APCs
(Figs. 1C 1F) . The results showed that the CD4 T cells responded to both stimuli, whereas the CD8 cells, although not responding to antigen, produced large amounts of cytokines when exposed to SEE, showing that they were not defective in inflammatory cytokine production and that, unlike CD4 IRBP-specific T cells, immunizing antigen alone was not sufficient to cause full activation of CD8 cells.