Figure 1.

Regulation of T cell IL-2 production by both B7-mediated costimulation and cell division. (a) Pooled BALB/c lymph node and spleen cells were labeled with CFSE, and T cells were stimulated in the presence of endogenous B7-mediated costimulation by the addition of soluble anti-CD3 antibody (1 μg/ml). (b) Alternatively, B7-mediated costimulation was blocked during primary activation by the addition of CTLA4Ig (15 μg/ml). After 4 days, cultures were washed, plated in fresh medium, and rested for an additional 48 hours. T cells were then restimulated for 5 hours by the addition of polystyrene beads (5 μm) coated with anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) antibodies, and the frequency of CD4⁺ cells producing IL-2 was assessed by flow cytometry. Plots show IL-2 production as a function of cell division (CFSE fluorescence) in the CD4⁺ T cell subset. Vertical lines delineate the divided from the undivided cells, and horizontal lines denote the maximal fluorescence of cells stained with isotype control antibody. Values in each corner represent the proportion of the CD4⁺ events that fall in each quadrant. Cells cultured in medium with no stimulus and stained with specific antibody were less than 0.1% positive for IL-2. These data are representative of three independent experiments. The frequency of IL-2 producers among CD3/CD28-primed T cells varied from experiment to experiment (range: ∼10–35%); however, the frequency of IL-2 producers in cultures primed in the presence of CTLA4Ig was consistently reduced two- to fourfold.