Figure 1.

Arginine N-methyltransferase inhibitor 1 (AMI-1) treatment increases the numbers and function of CD4⁺CD25⁺Foxp3⁺ Tregs in vitro. (A) Mouse splenocytes were stimulated with 1 µg/ml anti-CD3 and CD28. (B) Naïve CD4⁺ cells were differentiated into Tregs, and (C,D) FACS-sorted Tregs were stimulated with 100 µM AMI-1 or not (C) or 20 ng/ml IL-6 in the presence of 100 µM AMI-1 (D) under the stimulation with anti-CD3 and CD28 antibodies for 3 days and subjected to Foxp3 staining. The percentages of Foxp3-positive cells in the CD4⁺ T cell subset from representative subjects of three experiments were determined with flow cytometry. The data shown are means ± SEM in the right panel. (E,F) Tregs were treated with AMI-1 at 1–100 µM for 24 h and washed three times. The treated cells were then mixed with Teff cells at a 1:2 ratio. (E) The cells were analyzed for IFNγ after 48 h with FACS. (F) Cell proliferation was assayed with CFSE at day 4. The results are representative of three separate experiments.