Figure 2.

Examination of the effects of culture of naïve CD4⁺CD8⁻CD25⁺Treg with rIL-2 alone to culture with rIL-2 and alloantigen. To examine if the effects of culture were due to rIL-2 or alloantigen, we compared the phenotype of Treg after culture with rIL-2 alone, PVG alloantigen alone to that after culture with both rIL-2 and PVG alloantigen. Cultures for 4 days were carried out as described in Figure 1. (A,B) Staining for CD8α and CD8β was as for Figure 1. Culture of fresh tTreg with rIL-2 and no alloantigen also induced CD8α expression similar to when tTreg were cultured with rIL-2 and alloantigen. Again, no CD8α was induced by culture with PVG in the absence of rIL-2. There was a modest induction of CD8β with both rIL-2 and a combination of rIL2 and alloantigen stimulation. Data from two separate experiments and P values were calculated using a student t-test. (C,D) RT-PCR of tTreg after culture with rIL-2, PVG alloantigen and both rIL-2 and PVG alloantigen showed induction of mRNA for ifngr and Il12b2 in both tTreg cells cultured with rIL-2 alone and in tTreg cells cultured with both rIL-2 and PVG alloantigen, but not in tTreg cultured with no rIL-2 and PVG alloantigen.