CCR4 induction following in vitro CD3/CD28 stimulation. (a) Sorted CCR4^–CD4⁺ T cells purified from spleens of 16- to 18-week-old diabetic NOD mice were analyzed for expression of CCR4 and activation markers following in vitro CD3/CD28 stimulation. CCR4-negative cells were incubated in vitro for 2 days in the presence or absence of anti-CD3/anti-CD28 mAb’s. The majority of CCR4-negative populations induced CCR4 expression following stimulation with anti-CD3/anti-CD28 mAb’s. These CCR4-positive cells also expressed CD25 and high levels of CD44 and CD69. Open area, media stimulation; filled area, anti-CD3/anti-CD28 stimulation. (b) The culture supernatants generated from CD3/CD28-stimulated (black bars) and media-stimulated cells (white bars) were analyzed for IL-2, IFN-γ, and IL-4 by ELISA.