Functionality of CD28 molecules induced on CD4⁺CD28^null T-cell clones.

CD28 molecules induced on CD4⁺CD28^null T-cell clones are functional. CD4⁺CD28^null T-cell lines and clones were cultured with anti-CD3 mAb and IL-12 for 6 days to induce CD28 expression (Figure 4). Cells were then harvested and subsequently activated for 24 hours with anti-CD3 mAb or with anti-CD3 and anti-CD28 mAbs together. The cells were then analyzed by 4-color flow cytometry for the expression of the IL-2R α-chain (CD25) and CD40L. Histograms are shown for gated CD4⁺CD28⁺ and CD4⁺CD28^null cell fractions; results are representative of 5 experiments with different T-cell clones and lines. Costimulation with anti-CD3 and anti-CD28 mAbs (solid line), but not with anti-CD3 mAb alone (dotted line), resulted in up-regulation of CD25 and CD40L expression on cells that re-expressed CD28; however, expression of CD25 and CD40L was not induced on cells that failed to express CD28. In all histograms, the shaded areas represent staining with an isotype control.