Figure 4. Restrained infiltration of effector T cells and increased expression of CD25⁺Foxp3⁺ Tregs upon in vivo silencing of AIF1 in NOD mice.

Pancreata from treatment vs. control NOD mice were harvested for 3 or 9 weeks after initial injection of siRNA oligonucleotides targeting AIF1 (siAIF1) (n = 6) or scrambled controls (siScramble) (n = 6). Flow cytometric analysis was performed to assess changes in the CD45⁺TCRβ⁺CD4⁺ T helper population and changes in frequency of CD25⁺Foxp3⁺ Tregs (A) 3 weeks or (B) 9 weeks after in vivo AIF1 silencing of NOD mice. Singlets were interrogated by gating SSC-A vs. SSC-H (data not shown). (C) Percentage of CD25⁺Foxp3⁺ Tregs was assessed by flow cytometric analyses at 9-, 12-, 15-, 18-, and 24-week-old NOD mice (n = 2 of each time point of each group) after in vivo silencing for AIF1 vs. control oligonucleotides. For gene expression studies, pancreas cells were sorted for CD45⁺TCRβ⁺ cells before real-time PCR analyses (D) 3 weeks or (E) 9 weeks after in vivo AIF1 silencing of NOD mice. Statistical significance was determined by the 2-tailed Student’s unpaired t test. Data are shown as mean ± SEM of pooled samples of two independent experiments normalized to respective internal loading controls. *P < 0.05; **P < 0.01. AIF1, allograft inflammatory factor-1; ns, not significant; ND, not determined.