Fig. 4.

Met prevents the differentiation of naïve CD4⁺ CD25⁻ cells into CD4⁺ CD25⁺ Foxp3⁺ Treg cells. Naive CD4⁺ CD25⁻ cells purified from BALB/c spleen cells were labeled with CFSE and incubated for 6 h in the presence or absence of 10 μM Met.

(A) Met pretreatment of naïve CD4⁺ CD25⁻ cells did not affect the expression of CD25 or Foxp3. (B) Naive CD4⁺ CD25⁻ cells pretreated with or without Met as in (A) were stimulated for 3 days with the anti-CD3 mAb or anti-CD3 mAb + anti-CD28 mAb in the presence of graded doses of TGF-β, as indicated. Cell division and Foxp3 expression were monitored simultaneously. Foxp3 expression was significantly inhibited in cells pretreated with Met, as shown in the histograms. (C) The differentiated cells stimulated with anti-CD3 mAb + anti-CD28 mAb in the presence or absence of 5 ng/mL TGF-β as in (B) were expanded for an additional 2 days (day 5) in the presence of IL-2 or analyzed on day 3. The relationship between the number of divisions and the ratio of Foxp3⁺/Foxp3⁻ was plotted. No additional increase in the ratio of Foxp3⁺/Foxp3⁻ was observed with progressive division of cells pretreated with Met. (D) CD25 expression in Foxp3⁺ iTreg cells on days 3 and 5 (in C) was examined in parallel with cell division. The results in (A) to (D) are representative of four independent experiments.