Total RNA was isolated from nTregs or alloantigen induced Tregs (alloTregs), and
absolute levels of mRNA for Foxp3, CD25, TGF-β1, IL-2, IFN-γ,
IL-10, IL-4, or granzyme B were measured with the use of real-time quantitative
PCR assays. (A) Individual and mean (±SE) mRNA levels
are shown as a ratio of mRNA copies per one μg of RNA to 18S ribosomal
RNA copies per one pg of RNA. Levels of Foxp3 mRNA were not different between
alloTregs and nTregs (937 ± 104 vs. 976 ± 120, P>0.05) and
levels of CD25 mRNA (3606 ± 588 vs. 839 ± 150, P=0.0003) and
TGF-β1 mRNA (154 ± 32 vs. 58 ± 16, P=0.01) were higher in
alloTregs than nTregs. Levels IL-2 mRNA (0.45 ± 0.2 vs. 2.26 ±
1.0, P=0.01), IFN-γ mRNA (0.86 ± 0.5 vs. 9.64 ± 4.6,
P=0.007), IL-10 mRNA (0.44 ± 0.2 vs. 2.29 ± 0.6, P=0.01) and
granzyme B mRNA 3.05 ± 1.8 vs. 6.56 ± 1.2, P=0.02) were lower in
alloTregs than nTregs. (B) The ratio of Foxp3 mRNA to granzyme
B mRNA (1259 ± 331 vs. 199 ± 34, P=0.01) was higher in alloTregs
than nTregs. (C) The ratio of TGF-β1 to granzyme B mRNA
(182 ± 66 vs. 12 ± 5, P=0.0003) was higher in alloTregs than
nTregs. P values calculated using the Mann-Whitney test.